Analyte detection and apparatus therefor

ABSTRACT

The present invention relates to sensitive SE(R)RS based methods for detecting analytes such as explosives and drugs, which may be present in a sample at extremely low levels. The methods may be generally carried out in situ employing novel chemistry which is compatible with flow-cell technology and with time-scales and concentrations required for rapid and informative screening of large numbers of samples. The present invention also relates to novel compounds e.g. synthons and apparatus for use with the methods disclosed.

[0001] The present invention relates to sensitive methods for detectinganalytes such as explosives and drugs, which may be present in a sampleat extremely low levels. The methods may be generally carried out insitu employing novel chemistry which is compatible with flow-celltechnology and with time-scales and concentrations required for rapidand informative screening of large numbers of samples. The presentinvention also relates to novel compounds and apparatus for use with themethods disclosed.

[0002] The detection of plastic explosives, of drugs of abuse, oftherapeutic agents, and of environmental pollutants is a field ofgrowing importance in which there is a need for fast reliable, robustand simple detection methods. A major problem in this field is that therequired analyte is often present in very low concentrations in a matrixsuch as a vapour or a body fluid. Thus, detection methods that aresensitive and rapid and which identify the analyte from other speciesare required.

[0003] One method of detection employs the detection of Ramanscattering. Briefly, Raman scattering occurs when a light sourceirradiates a sample and scattered light is given off. Most of the lightis scattered with the same frequency as that of the incident light but aweak component is scattered one vibrational unit different. The weakcomponent is Raman scattering. By subtracting the frequency of the Ramanscattered light from the frequency of the incident light, a vibrationalspectrum characteristic of the molecule can be obtained. The light canthen be detected in a suitable spectrometer, many of which arecommercially available. The detection of Raman scattering is attractivesince it uses visible or near infrared radiation to provide theexitation. Moreover, flexible and effective optics can be designed andwater gives a weak signal so that detection in aqueous solution ispossible. Further, the set of signals obtained gives a unique patternfrom which a particular analyte can be identified. However, the maindisadvantage of Raman scattering is that it is not sufficientlysensitive and is not therefore generally suitable for detecting analytesat extremely low concentrations, and fluorescence can interfere withdetection.

[0004] The sensitivity of Raman scattering may however be improved.Firstly, if the analyte is adsorbed onto a suitably roughened metalsurface of which silver and gold are the most widely used, then there isan interaction between the analyte and the surface electron waves on themetal (plasmons) which provide an enhancement in the intensity of theRaman scattering by a factor claimed to be 10⁶. This technique is knownas surface enhanced Raman scattering (SERS).

[0005] Another method of enhancing sensitivity is to use a dye with anabsorption maximum at or close to the frequency of the excitingradiation. This enhanced scattering, called resonance Raman scattering,provides an increase in sensitivity of a few orders of magnitude inideal cases. However, it is possible that fluorescence will interferewith this process.

[0006] Combining SERS and resonance Raman scattering: to give surfaceenhanced resonance Raman scattering (SERRS), provides more sensitivityand the conditions under which single molecule detection has beenclaimed. SERRS has been previously described, see for example Rodger, C.H., Smith, W. E., Dieht, G., Edmonson, M., J. Chem. Soc. Dalton Trans.,1996, 5, 791 and references therein to which the reader is directed forbackground information. Surprisingly, there is a widespread fluorescencequenching mechanism on the silver surface. This means that almost alldyes give SERRS rather than fluorescence on the surface enabling the useof more extensive derivatisation chemistry than is possible byfluorescence. Further, the SERRS active material scatters so stronglythat the signal can be picked out from the background without the needfor the removal of the matrix in which the sample is present so thatseparation procedures either before or after analysis are often notrequired.

[0007] However, the main disadvantage of SERRS is that it requires aspecially labelled dye. To obtain reproducible results, this dye mustalso adhere strongly to a metal surface. Thus, although it is relativelyeasy to obtain very low atomolar detection limits, very variable resultsare often obtained and many molecules for which sensitive analysis wouldbe of value are precluded from the method since they are not colouredand do not stick to the surface.

[0008] Recent patents describe the use of SERRS in DNA detection and inantibody detection (WO97/05280 & PCT/GB99/00588 respectively). In boththese cases, a pre-formed molecule is used as the actual analyte. In DNAchemistry this is a label and in antibody chemistry it is a labelledantigen or ligand which is displaced from the antibody.

[0009] However, it will be appreciated that detection of analytes, suchas explosives or drugs is not generally possible because of the lack ofa suitable chromophore and/or the ability of the analyte to adhere to ametal surface. Moreover, it is desirable that detection of explosives ordrugs can be effected quickly, for practical reasons, which is notgenerally possible using existing techniques.

[0010] It is an object of the present invention to obviate and/ormitigate at least one of the aforementioned disadvantages.

[0011] According to a first aspect the present invention provides amethod for detecting an analyte in a sample, using surface enhanced(resonance) Raman scattering (SE(R)RS) detection, comprising the stepsof:

[0012] a) mixing the sample with a reagent such that any analyte presentin the sample reacts with the reagent thereby forming a derivatisedanalyte, wherein the derivatised analyte comprises a chromophore;

[0013] b) mixing said derivatised analyte with a SE(R)RS activesubstrate so as to adhere the derivatised analyte thereto; and

[0014] c) detecting the derivatised analyte by way of SE(R)RS detectionwhereby any derivatised analyte detected may be correlated with analytepresent in the sample.

[0015] It is to be appreciated that SER(R)RS refers to SERS or SERRSdetection, with SERRS being preferred.

[0016] Examples of analytes which may be detected include, aldehydes,amines, explosives, drugs of abuse, therapeutic agents, metabolites andenvironmental pollutants. The sample may be any suitable preparation inwhich the target analyte is likely to be found. However, convenientlythe sample may be in solution or transferred to a solution beforereacting with the reagent. Thus, for example when detecting explosivesor drugs of abuse, a sample of air or breath respectively, may be takenand any target analyte absorbed onto a suitable substrate. Thereafter,any target analyte may be removed from the substrate by washing with asuitable solvent, such as dimethyl formamide (DMF), acetone ortetrahydrofuran (THF).

[0017] For example in the determination of TNT or RDX from the vapourphase, the vapour can first be collected on a suitable material such astenax and a small amount of solvent washed through the material toproduce a small amount of explosive in solution. The preferred solventfor this purpose is dimethyl formamide.

[0018] For effective SERRS analysis, a chromophore of a suitablewavelength to be in resonance with the laser chosen, must be present inthe analyte or a chromophore must be created by derivatisation of theanalyte before analysis. Moreover in either case effective adsorption tothe surface must be achieved. Thus, the reagent which is used toderivatise the analyte, may provide a chromophore, may provide incombination with the analyte, a chromophore and/or render the analytesusceptible to adhering to the SERRS active substrate.

[0019] In some instances simple derivatisation of the analyte with thereagent may not be possible. In such instances it may first, benecessary to carry out chemical functionalisation such as a reduction oroxidation of the analyte prior to reacting with the reagent. Moreover,it is often the case that the analyte to be detected will not contain asuitable chromophore or be able to adhere to the SERRS active metalsubstrate. In this manner the reagent reacts with the analyte forming aderivatised analyte possessing a suitable chromophore and the ability toadhere to a SERRS active metal substrate. Alternatively reaction withthe reagent may generate a derivatised analyte with a chromophore. Thederivatised analyte may then be adhered to a SERRS active metalsubstrate by way of an aggregating agent.

[0020] Examples of these two separate approaches of achieving this willbe described in detail herein. In the first a colourless analyte isreacted with a reagent to provide a derivatised molecule with achromophore and adheres to the SERRS active substrate using a suitableaggregating agent. The preferred method uses a new synthon specificallydesigned for SERRS, which reacts rapidly with the analyte, providing achromophore and a group for attaching to the SERRS active surface.

[0021] For TNT the use of a standard chemical reaction may be used, theJanowsky reaction, to provide a coloured species (ie. a molecule with achromophore in the visible region). This provides a chromophore but doesnot provide optional surface attachment. It has been found that usingcertain aggregating agents required in any case to aggregate the colloidfor the best effect that the coloured species can be incorporated intothe aggregated colloid. The preferred aggregating agent for this purposeis poly-L-lysine. This reaction is effective for TNT down to aconcentration of about 10⁻¹⁰ molar. This corresponds to 2 pg of materialin the sample. This number depends entirely on the method of SERRSdetection used. Surprisingly this procedure will also work with acid butnot sodium chloride aggregation.

[0022] TNT may also be derivatised by a reduction process which producesan azo chromophore from TNT. This procedure is also rapid and providessuperior detection limits.

[0023] Surface enhancement may also be improved in the Janowsky reactionby utilisation of a surface seeking ketone/aldehyde in the reaction withTNT. Ketones/aldehydes of generic structure (A) were prepared bydiazotisation of an appropriate amino acetophenone/aldehyde derivativeand coupling to a suitable benzotriazole or 8-hydroxyquinolinederivative. Ketones/aldehydes of generic structure (B) were preparedfrom diazotisation of 5-aminobenzotriazole (or derivatives of the 5ABTgeneric structure) and coupling with suitable aromaticketones/aldehydes. Any other aromatic or aliphatic ketone/aldehyde maybe used in this reaction to form the Janowsky complex (C). Examplesinclude the following where R=any reactive functional group such asamine, nitro, vinyl, formyl, acetyl, amide, azo, imine, alkyl, phenyl,halide, trifluoromethyl, acid, ester, ether, diazonium, naphthyl, aryl,alkenyl, cycloalkyl, thiol, nitroso, phenol, hydroxylamine, maleimide,succinimide, imide, heterocyclic, nitrile, diazo, acyl, azide etc. . .and where compounds with multiple functional groups where R can be anyof the groups above in any combination.

[0024] TNT may also be reduced using suitable agents such as thosedescribed for RDX (see later) to yield primary aromatic amines. Theseamines can be diazotised and reacted with a suitable coupling agent toyield highly coloured azo dyes, which display unique and selective SERRSspectra.

[0025] This reaction is successful using the flow cell methodologydescribed herein, where it was shown that TNT can be derivatised anddetected in-situ.

[0026] A second and preferred approach developed by the presentinventors for SERRS detection involves the creation of special SERRS“synthons”. (A synthon is a molecule specifically designed as a buildingblock which coupled with other reagents, enables a range of chemistry tobe carried out simply).

[0027] According to this procedure, the present inventors havesynthesised novel molecules which have a group which adheres strongly toa SERRS active substrate such as a silver or gold colloidal surface andwhich also has a group which is reactive to or may complex with specificanalytes. In some cases, the chromophore is formed when the synthonreacts with the analyte. In others, the synthon already contains achromophore and reaction at the analyte only alters the nature of thechromophore, thus altering the SERRS signals. Surface adhering groupssuitable for a SERRS active metal substrate can be used. Alternatively,polymerisation reactions such as those obtained with polyphenols whichcan incorporate a molecule in a polymer on the surface could also beused.

[0028] Examples of suitable synthons include

[0029] wherein X may be substituted on one or more positions of thearomatic ring and is an amine, amide, aldehyde, thiol, diazo group,nitro, a vinyl group or other active group. For example, the benzenering may be substituted at two positions such as the 5 & 6, 5 & 7 and 4& 7 positions. In particular X may be: —NH₂; —R—CONH₂, H₂NCO—R—CONH₂, or

[0030] —CHO wherein R is C₁-C₄ alkyl or alkenyl; a diazonium halide, ora mono, di or tri nitro phenyl. Examples include those shown below andat the end of the description:

[0031] However these molecules do not generally contain a chromophorewith an absorption band in the visible or near infrared region. Sincevisible or near infrared excitation is preferred for SERRS detection, itis necessary that a chromophore is formed during the derivatisationreaction or that the analyte is itself a chromophore. Moreover, sincebenzotriazole chemistry can affect the nature of substitution reactions,it can be beneficial to put a spacing group between the benzotriazoleand the “X” ligands. Thus, very similar synthons can be made as shownbelow and at the end of the description:

[0032] where X has the same meaning as defined previously, and Y may bean alkyl, aryl, alkenyl, alkynyl, cycloalkyl group including heretoderivatives of the preceding groups, or Y may be any atom that canprovide two or more bonds to link the two groups together eg. O or B.Most preferably Y will be an amine, imine or azo linkage.

[0033] Additionally, synthons can be created in which the dye is placedbetween the benzotriazole group and the functional group as below and atthe end of the description:

[0034] wherein X has the same meaning as defined previously.

[0035] The present invention therefore also provides novel synthons foruse in the methods of detection described herein.

[0036] Example 7 illustrates the use of one of the aforementionedsynthons in the SERRS detection of a molecule containing a chromophore.For example, a haem with a sulphydryl group in the same molecule willreact with this synthon to form a coloured compound which adheresstrongly the synthon to the metal surface.

[0037] This type of reaction may be used to assay for example proteinscontaining a thiol and a chromophore such as a haem. Example 7 showsthat this reaction is successful for haemoglobin. Haemoglobin does notdirectly produce intense SERRS without the reaction with the synthonsince it does not adsorb on the silver surface. Other groups for exampleamines such as terminal lysine can be coupled for example using abenzotriazole aldehyde.

[0038] As mentioned above the synthons may comprise an aldehyde, amine,thiol or other reactive group as well as a surface complexing group.These may be useful for example for the detection of amines andaldehydes in the environment. For amines or aldehydes, a number ofSchiff base compounds can be formed. Here the azomethine group formed inthe reaction creates a chromophore.

[0039] A specifically important example is example 8 where a synthon isused to determine the presence in a sample of the plastic explosive RDX.RDX is particularly difficult to analyse because any alteration in themolecule to form a chromophore can lead to its decomposition. Controlledreduction of RDX has been achieved using any reducing system as shown,for example, in the table below. Two preferred ways were chosen; theproducts of which were characterised fully by conventional spectroscopictechniques (NMR mass spectrometry, microanalysis, IR, etc.). Bothmethods of derivatisation allow the sensitive and selectivedetermination of RDX and RDX reduction products by SERS and SERRS atultra-low concentrations. TABLE 1 Example of RDX Reducing AgentsReduction Systems Reduction Systems CrCl₂/DMF hydrazine/graphite orRh(C) Na/Hg Zn/AcOH BF₃-etherate NaBH₄/Pd-C NaBH₄/AcOH BER-Ni(OAc)₂Zn/DMF H₂/Pd(C) Indium/NH₄Cl SmI₂ SnCl₂ Fe LiAlH₄ Sulphide Formate/Pd(C)TiCl₃

[0040] The first method relates to the reduction of nitramine. Forexample a hydrazine derivative is obtained by reduction with Na/Hg,Zn/AcOH or any other suitable reducing agent, which is then reacted withan aldehyde or ketone to produce a coloured Schiffs base which itselfmay be further derivatised. Alternatively hexamine is formed byreduction of RDX with metal hydrides and reacted with any diazonium saltto yield an azo dye. Additionally, catalytic hydrogenation over Pdmaintains the RDX ring structure to afford the mono, di and trisubstituted RDX hydrazine derivatives—see below.

[0041] Furthermore RDX can be deprotonated by a suitable base to yieldseveral species, which may be reacted further to yield products suitablefor SERRS analysis.

[0042] It may be deprotonated to an anion, which can be quenched with asuitable electrophile such as benzotriazole aldehyde to yield a hydroxylderivative. It may also expel HNO₂ to yield an unstable imine, which mayfor example be reduced to an amine or reacted with a diene to give aDiel's-Alder additional product—see below:

[0043] Both methods provide the basis for an assay sufficientlysensitive and selective to use the same collection techniques asdescribed earlier for TNT and analyse RDX for the vapour phase.

[0044] Another important explosive which may be detected using themethods of the present invention is PETN. PETN may be reduced orhydroysed to yield an alcohol, which for example can be further reactedto yield a Schiff's base compound incorporating benzotriazole, thusfacilitating detection by SERRS. Compound (3) as shown below and it'sderivatives may also be reacted in other ways to produce molecules whichare suitable for direct SERRS analysis or further derivatisation.

[0045] Any other nitro, nitramine or nitrate ester containing molecule,whether they are explosives or not may be derivatised by the methodsdescribed above.

[0046] It should be appreciated that the present invention provides forthe first time methodology which is suitable for in situ chemistry andSERRS detection. That is, apparatus may be designed, as will beexemplified below, which allows a sample to be added to the apparatus,the chemical reactions carried out therein and SERRS detection done, allwithin a single apparatus and preferably within a matter of minutes.Preferably the time taken from obtaining the sample to generating aSERRS spectrum should be less than one minute preferably less than 20seconds.

[0047] Moreover, the speed and sensitivity with which SERRS detectioncan take place enables the detection of derivatised analytes which arenot typically stable for any length of time. Thus, the chemistry taughtherein does not have to provide a stable derivatised analyte. Thederivatised analyte has only to be stable long enough for detection totake place. Thus, the derivatised analyte many only have to be stablefor a few minutes, for example 1-2 minutes, or even for a few seconds.

[0048] The SERRS-active surface may be any suitable surface, usuallymetallic, which gives rise to enhancement of the Raman effect, of whichmany are known from the SERRS literature. It may for instance be anetched or otherwise roughened metallic surface, a metal sol or, morepreferably, an aggregation of metal colloid particles. Silver, gold orcopper surfaces, especially silver, are particularly preferred for usein the present invention and again, aggregated colloid surfaces arebelieved to provide the best SER(R)S effect.

[0049] The surface may be a naked metal or may comprise a metal oxidelayer on a metal surface. It may include an organic coating such as ofcitrate or of a suitable polymer, such as polylysine or polyphenol, toincrease its sorptive capacity.

[0050] Where the surface is colloidal, the colloid particles arepreferably aggregated in a controlled manner so as to be of a uniformand desired size and shape and as stable as possible againstself-aggregation. Processes for preparing such unaggregated colloids arealready known. They involve, for instance, the reduction of a metal salt(eg. silver nitrate) with a reducing agent such as citrate, to form astable microcrystalline suspension (see P. C. Lee & D. Meisel, J. Phys.Chem. (1982), 86, p3391). This “stock” suspension is then aggregatedimmediately prior to use. Suitable aggregating agents include acids (eg.HNO₃ or ascorbic acid), polyamines (eg. polylysine, spermine,spermidine, 1,4-diaminopiperazine, diethylenetriamine,N-(2-aminoethyl)-1,3-propanediamine, triethylenetetramine andtetraethylenepentamine) and inorganic activating ions such as C1⁻, I⁻,Na⁺ or Mg²⁺. To increase control over the process, all equipment usedshould be scrupulously clean, and reagents should be of a high grade.Since the aggregated colloids are relatively unstable to precipitation,they are ideally formed in situ with the detection sample and the SERRSspectrum obtained shortly afterwards (preferably within about 15 to 30minutes of aggregation).

[0051] Ideally, a material such as spermine or spermidine is introducedto assist control of the aggregation process. The aggregation may becarried out at the same time as, or shortly after, the surface isintroduced to the other species in the detection sample.

[0052] The colloid particles are preferably monodisperse in nature andcan be of any size so long as they give rise to a SERRS effect—generallythey will be about 4-50 nm in diameter, preferably 25-36 nm, though thiswill depend on the type of metal.

[0053] Preferably, the surface comprises silver colloid particles, whichare preferably substantially hexagonal in shape and of about 20-36 nmmaximum diameter.

[0054] Adhering the derivatised analyte with the SERRS-active surfacewill typically be by chemi-sorotion of the complex onto the surface, orby chemical bonding (covalent, chelating, etc.) of the complex witheither the surface or a coating on the surface, either directly orthrough a linking group. The association will usually be via suitablefunctional groups on the derivatised analyte, such as charged polargroups (eg. NH₃ ⁺ or CO₂), attracted to the surface or surface coating(eg. to free amine groups in a polyamine coating). Clearly, the type ofassociation will depend on the nature of the surface and the label inany given case; different functional groups will be attracted to apositively-charged surface, for instance, as to a negatively-chargedone.

[0055] Suitable groups by which the complex may be bound to the activesurface include complexing groups such as nitrogen, oxygen, sulphur andphosphorous donors; chelating groups; bridging ligands and polymerforming ligands. Specific details of preferred methods of adhering thederivatised analyte with the SERRS active substrate are described inWO97/05280.

[0056] The method for obtaining the SERRS spectrum, once the derivatisedanalyte has been adhered to the metal substrate, may be conventional.The present invention is generally concerned with chemical modificationsto existing SERRS techniques, ie. modifications to an analyte, to makethe analyte viable for use in detection by SERRS.

[0057] By way of example, however, the following might apply to thespectroscopic measurements:

[0058] Typically, the methods of the invention will be carried out usingincident light from a laser, having a frequency in the visible spectrumie. 380 nm-780 nm, particularly between 400 nm-650 nm (the exactfrequency chosen will generally depend on the chromophore used in eachcase—frequencies in the red area of the visible spectrum tend, on thewhole, to give rise to better surface enhancement effects). However, itis possible to envisage situations in which other frequencies, forinstance in the ultraviolet (ie. 200 nm-400 nm) or the near-infraredranges (700 nm-100 nm), might be used. Thus, SERRS detection may beconducted between about 300 nm-1100 nm.

[0059] The selection and, if necessary, tuning of an appropriate lightsource, with an appropriate frequency and power, will be well within thecapabilities of one of ordinary skill in the art, particularly onreferring to the available SERRS literature. To achieve highly sensitivedetection, using SERRS, a coherent light source is needed with afrequency at or close to the absorption maximum for the chromophore (asdescribed above) or that of the surface plasmons. If lower sensitivitiesare required, the light source need not be coherent or of high intensityand so lamps may be used in combination with a monochromator grating orprism to select an appropriate excitation frequency; here, there is noneed to operate at the resonant frequency of the chromophore or theplasmons.

[0060] The source can be used to excite the chromophore directly on anactive surface such as an electrode; by shining through a SERRS-activecolloidal suspension; or by means of evanescent waves via a waveguidecoated with a SERRS-active surface.

[0061] The light can be conducted from the source to the active surfaceby reflection in mirrors and can be focussed to give a higher light fluxby passing through lenses. A suitable apparatus for SERRS analyses is afluorescence microscope with signal detection at 90° to the excitationbeam. A fluorescence microscope with confocal optics is alsoappropriate. The use of microscope optics permits very small areas orvolumes to be analysed.

[0062] The light can alternatively be conducted from the source to theactive surface through a waveguide. This gives flexibility as to thesite of sampling; the waveguide can be scanned over the active surfaceor dipped into a SERRS-active colloid suspension. A waveguide isparticularly appropriate for use in analyses carried out in the solutionphase for example in the wells of a microtitre plate. The waveguide canbe carried on a robot arm and deposited sequentially in each well forhigh throughput screening of many samples.

[0063] A waveguide coated with a SERRS-active surface may also be usedselectively to detect analytes which bind to that surface. The principleis as follows. Light is passed along a waveguide by total internalreflection. However, molecules closely bound to the external surface ofthe waveguide may still be excited by the electric field of the light(“evanescence”). Emissions, such as SERRS emissions, resulting from thisexcitation pass on through the waveguide and can be detected at itsoutput end.

[0064] In SERRS the primary measurements are of the intensity of thescattered light and the wavelengths of the emissions. Neither the angleof the incident beam nor the position of the detector is critical. Withflat surfaces an incident laser beam is often positioned to strike thesurface at an angle of 600 with detection at either 90° or 180° to theincident beam. With colloidal suspensions detection can be at any angleto the incident beam, 90° again often being employed.

[0065] The intensity of the Raman signals needs to be measured againstan intense background from the excitation beam and for this reason theuse of Raman analytes with large Stokes' shifts is an advantage. Thebackground is primarily Raleigh scattered light and specular reflection,which can be selectively removed with high efficiency optical filters.

[0066] Several devices are suitable for collecting SERRS signals,including wavelength selective mirrors, holographic optical elements forscattered light detection and fibre-optic waveguides. The intensity of aSERRS signal can be measured for example using a charge coupled device(CCD), a silicon photodiode, or photomultiplier tubes arranged eithersingly or in series for cascade amplification of the signal. Photoncounting electronics can be used for sensitive detection. The choice ofdetector will largely depend on the sensitivity of detection required tocarry out a particular assay.

[0067] Note that the methods of the invention may involve eitherobtaining a full SERRS spectrum across a range of wavelengths, orselecting a peak and scanning only at the wavelength of that peak (ie.Raman “imaging”).

[0068] Apparatus for obtaining and/or analysing a SERRS spectrum willalmost certainly include some form of data processor such as a computer.

[0069] Raman signals consist of a series of discrete spectral lines ofvarying intensity. The frequencies and the relative intensities of thelines are specific to the derivatised analyte being detected and theRaman signal is therefore a “fingerprint” of the derivatised analyte. Ifa SERRS analyser is being used selectively to detect one analyte out ofa mixture then it will be necessary to detect the entire “fingerprint”spectrum for identification purposes. However if the analyser is beingused to quantitate the detection of one or several analytes, each ofwhich has a unique spectral line, then it will only be necessary todetect signal intensity at a chosen spectral line frequency orfrequencies, or to detect all Raman scattering using a filter to excludeRalleigh scattering.

[0070] Once the SERRS signal has been captured by an appropriatedetector, its frequency and intensity data will typically be passed to acomputer for analysis. Either the fingerprint Raman spectrum will becompared to reference spectra for identification of the detected Ramanactive compound or the signal intensity at the measured frequencies willbe used to calculate the amount of Raman active compound detected.

[0071] A commercial SERRS analyser of use in carrying out the inventionwould be expected to consist of the following components: a laser lightsource, the appropriate optics for carrying the light to the SERRSactive surface, a stage for mounting the sample for analysis, optics forreceiving the Raman signal, a detector for converting the Raman signalinto a series of intensities at certain wavelengths and a data processorfor interpreting the wavelength/intensity data and providing ananalytical output.

[0072] The light source, optics, detector and processor have alreadybeen referred to. The stage for mounting the sample could be designed toaccommodate one or more of the following solid supports: a microscopeslide or other flat surface such as a silicon wafer or chip, amicrotitre plate or a higher density array microwell plate, a capillary,a flow-cell or the like or a membrane.

[0073] An assay could be carried out on a solid support and the supportinserted into a SERRS reader for analysis. Alternatively the assay couldbe carried out in a separate vessel with a subsequent transfer of theassay components to the solid support for inserting into the analyser.The use of robotics to transfer solid supports to and from a SERRSanalyser stage would permit the development of a high throughput systemwithout significant operator input with samples being run and analysedautomatically.

[0074] A particularly preferred type of assay would involve the use offlow-cell technology, as will be described below.

[0075] To release the potential for SERRS for rapid detection, thepresent inventors have designed a flow cell compatible with thechemistry described herein. Additionally ways of providing colloidwithout requiring to pre-prepare it have been developed as well aspre-prepared colloid which remains stable over time (eg. 3-6 months).With this combination, a head can be treated which can be attached to aRaman spectrometer. The unit will require only standard chemicals and/orthe specially designed synthons and the same device will be capable ofuse for a wide range of sensitive and selective analytical procedures.

[0076] An example of a suitable flow cell system is shown in FIG. 1. Ascan be seen the flow cell system (10) comprises three reservoirs (12, 14and 16) which contain the reagent, colloid comprising the SERRS activesubstrate and aggregating agent, respectively. A pump (18) is providedto transfer the various solutions along tubing (20).

[0077] Sampling of the explosives may be from the vapour phase. To dothis, air may be sucked through a small tube containing a suitable,adsorbent material. The commercial material Tenax is suitable for thispurpose. An alternative method is to bubble the vapour through asuitable solvent such as acetone or DMF in which the explosive issoluble. In the case of the adsorbed sample a small amount of acetone orDMF or other suitable solvent is then injected through the sample anddissolves off the explosive. The solution is then passed into the flowcell. In the case of bubbling through the solvent, this sample will beused directly.

[0078] In use the collected sample then is added to the flow cell system(10) at the position represented by the arrow (A). The sample is thenpumped through the tubing (20) and mixed with the reagent by passingthrough mixing coil (22), such that derivatisation of the sample cantake place. By appropriate controls (not shown) the colloid isaggregated by passing the colloid through mixing coil (24) where it ismixed with the aggregating agent. The derivatised sample is then mixedwith the appropriate aggregated colloid in mixing coil (26) such thatthe derivatised analyte adheres to the aggregated colloid therebyenabling SERRS detection. The adhered analyte is then passed through acapillary tube (30) where SERRS is detected by way of an appropriatelytuned laser (32) and spectrophotometer (34).

[0079] Thus, in a further aspect the present invention provides adetection device for detecting the presence of an analyte in a sample byway of SE(R)RS, the device comprising at least one flow cell forcombining in-situ the sample to be analysed, a reagent capable ofreacting with any analyte present in the sample in order to provide aderivatised analyte comprising a chromophore, and thereafter reactingwith a SE(R)RS active substrate so as to adhere the derivatised analytethereto and detecting the derivatised analyte by way of SE(R)RS.

[0080] A key feature of the invention is that the chemistry iscompatible with the production of the colloid in situ in the flow cell.The advantage of this procedure is that the need for colloid as areagent is eliminated from the experiment and all colloid is preparedfresh. However, it was discovered that by the addition of citratethrough a side arm into the flow cell, stable colloid was created. Sincethe flow cell can be maintained continuously in use, a reproduciblecolloid product is obtained whereas with the standard batch processthere is very considerable interbatch variations. Thus, by correct useof the flow cell and the addition of stabilising agents such as citrate,a stock of colloid can be built up for use where analysis requirespreformed colloidal suspensions. An example of a method that willrequire this colloid is given immediately below.

[0081] Alternatively a small amount of colloid may be added to a surfaceand a SERRS analyte added thereto. This can give extremely largesignals. One of the advantages about this is that almost all the sampleis under the microscope beam all the time giving the maximum opportunityfor signal accumulation. This type of process can be adapted either towork with a moving xyz stage or with a robot arm, which depositsindividual components, under the microscope. This method is alsocompatible with hand-held devices.

[0082] As an example, a microdot of colloid may be added to a substratesuch as filter paper, silica, glass or metal. To this microdot, is addedthe SERRS active substance to be detected. Detection is then carried outusing a standard microscope or fibre optic probe. The preparation of theSERRS active material can either occur prior to addition to thesubstrate, on, addition to the substrate and silver or the order can bereversed and the chemistry carried out on the surface and the colloidadded subsequently. The use of microscale samples means that all of theavailable molecules are present under the microscope beam and are sensedat any one time.

[0083] An additional way of doing this is to use a microelectrode ofsilver suitably roughened. The electrode surface is then a very goodSERRS active substrate that can be regenerated in an electro chemicalcycle. Additionally, the size of the electrode can be controlled tomatch that of the area interrogated under the microscope or fibre opticprobe.

[0084] Examples of how this may be applied to detecting TNT are asfollows:

[0085] 10 microlitres of a solution of TNT in organic solvent is addedto a silica, alumina or filter paper surface. A solution of sodiumborohydride is then added with a micropipette to the drop. To thismixture is added a SERRS synthon, preferably benzotriazole aldehyde.Once the SERRS active Schiffs base compound has been formed, a microdotof silver colloid is applied. This is then interrogated under the Ramanspectrometer. To prepare the microdot of colloid, the colloid is spundown in the centrifuge. The colloid can then be resuspended in asuitable medium, such as a viscous organic solvent, an ink medium orgellatine. This prevents or controls spreading of the colloid. The sizeof the spot to be sampled is defined by the optics used. In thisexample, a×5 objective on a microscope was used.

[0086] Alternatively a suspension of the colloid centrifuged to increasesilver particle concentration may be applied using a micropipette to asilica surface. Using a microcuvette and a pipette, a small sample ofTNT in organic solvent is added to the microtiter plate well. To thiswell, is added a solution of borohydride. Following reduction of thenitro group to an amine, the SERRS synthon in this case either theazo-dye or the aldehyde is added to the microtiter plate well. Once thecolour has developed an aliquot from this well is then added to thesilver particles on the silica substrate. This sample is theninterrogated under the microscope.

[0087] Since in situ chemistry is carried out, the rate at whichreagents are added, the temperature, the efficiency of mixing and thepressure in the tubes requires to be carefully controlled. Initiallythis can be done by varying the size of the mixing coils and flow rates.To obtain more control, small pumps and valves can be added to theequipment. By encasing the flowcell itself in a metal block, it ispossible to use for example small heaters and/or Peltier coolers toachieve temperature control. Further mixing and dissolution can be aidedby placing the coils in an ultrasonic bath and faster heating may beobtained using for example a microwave generator.

[0088] Since rapid analyte detection is generally required, completereaction of all the available analyte may not be necessary but since theflow cell controls the time of mixing for both the aggregation procedureand the derivatisation reaction, results with good relative standarddeviations of about 1% are routinely obtained. Detection may be madethrough a conventional microscope lens focused on part of the capillaryfrom the run out from the flow cells. To enhance the sensitivity of thesystem further the capillary can be encased in a reflecting sphere sothat a large fraction of the scattered light is focused back onto thelens of the microscope and collected.

[0089] It is also known that the production of silver colloid isnotoriously unreliable. For example in the form of colloid productionpreferred here a standard reduction of silver nitrate by citrate iscarried out. The exact form of the colloid depends on heating rates,length of incubation time, stirring rates and other features.

[0090] It is possible to use a previously reported approach of usingpre-formed colloid (Cabalin, L. M.; Ruperez, A.; Lasemna, J. J. Analytica Chimica Acta, 1996, 318, 2, 203-210) and adding it to the flowcell as required. However, as mentioned above the present inventors havediscovered methods to produce colloid in situ using the flowcelltechnique which provide much more reproducible results and overcome theinter batch problem. In essence, a suitable reducing agent(cyano-borohydride) is mixed with the silver nitrate in the first coiland the colloid is produced in seconds. Since the rate of addition andtemperature can be controlled, this colloid is reproducible. Further,since the same form of colloid is provided on each occasion, much of theunreliability associated with SER(R)S has been removed. The importantpoint is that kinetic control of colloid production is obtained at thedetection point and the colloid need be stable only for a short time.However, it is also possible to reproducibly produce stable colloid.

[0091] Thus, in a further aspect the present invention provides a methodof preparing colloid for use in SER(R)S analysis, comprising the stepsof mixing a metal nitrate solution such as silver nitrate with areducing agent, such as cyano-borohydride, using a flow cell in order toform a colloid and thereafter adding citrate in order to stabilise thecolloid. Colloid produced in this manner has been observed to remainstable for at least 3 months.

[0092] The present invention will now be described in more detail by wayof example only and with reference to the following figures in which:

[0093]FIG. 1 shows a somewhat schematical representation of a flow cellaccording to the present invention;

[0094]FIG. 2 shows a design of flow cell used to detect TNT in a sample;

[0095]FIG. 3 shows the SERRS spectrum of a TNA/Janowsky complex oncolloid preaggregated by polylysine;

[0096]FIG. 4 shows the SERRS spectra of the TNT/Janowsky complex formedfrom 10⁻³M TNT in acetone on colloid aggregated with differentaggregating agents (-, 10 μl 0.01% polylysine; - - - , 10 μl 1% HNO₃; -20 μl 10% NaNO₃ (intensity ×10));

[0097]FIG. 5 shows SERRS intensity against time for 10⁻³M TNT/Janowskycomplex in 1 ml colloid; □, 5 μl 0.01% polylysine;, ∘10 μl polylysine; ▴20 μl polylysine;

[0098]FIG. 6 shows the SERRS spectrum from dye (9) at 10⁻⁹M;

[0099]FIG. 7 shows the SERRS spectrum of dye (10) at ˜10⁻⁴M;

[0100]FIG. 8a shows the SERRS spectra of dyes (14) and (15), both 514 mmand 10 secs;

[0101]FIG. 8b shows the x-ray tructure of dye (14);

[0102]FIG. 9 shows the SERRS spectrum of imine formed from TAT;

[0103]FIG. 10 shows the SERRS spectrum of5-carboxaldehyde-1H-benzotriazole;

[0104]FIG. 11 shows the SERRS spectrum of1H-benzotriazole-2,4-dinitrophenylhydrazone;

[0105]FIG. 12 shows the SERRS spectrum TNT derivatised by5-methyl-thiophenecarboxyaldehyde;

[0106]FIG. 11 shows the SERRS spectrum of TNT derivatised by5-carboxyaldehyde-1H-benzotriazole;

[0107]FIG. 14 shows a SERRS spectrum of horse hemoglobin at 5×10⁻⁵M=1500 μg of protein;

[0108]FIG. 15 shows a SERRS spectrum of benzotriazole maleimide coupledhemoglobin at 9.8×10⁻⁶M=29 μg;

[0109]FIG. 16 shows a SERRS spectrum of pyridine azine;

[0110]FIG. 16 shows a ¹H NMR study of the reduction of RDX by BER-Ni(Oac)₂;

[0111]FIG. 18 shows a SERRS spectrum of hexamine diazo;

[0112]FIG. 19 shows the UV-vis adsorption spectrum of 4-acetylpyridineJanowsky complex vs. acetone complex;

[0113]FIG. 20 shows the SERRS spectrum of 4-acetylpyridine Janowskycomplex (10⁻⁵ M);

[0114]FIG. 21 shows the UV-vis of Janowsky complex of BT azo ketone andTNT

[0115]FIG. 22 shows a diagram of a coil used for batch production ofborohydride reduced silver colloid;

[0116]FIG. 23 shows a diagram of a flow cell system for azoderivatisation and detection of TNT including on-line formation ofborohydride-reduced silver colloid;

[0117]FIG. 24 shows the reaction scheme for the formation of5-(2-methyl-3,5-dinitro-phenylazo)quinolin-8-ol from TNT in the flowsystem; and

[0118]FIG. 25 shows the SERRS spectrum of5-(2-methyl-3,5-dinitro-phenylazo)quinolin-8-ol prepared from 1.1×10⁻³ gTNT in the flow system.

[0119] All chemicals used were of laboratory grade. Tetrahydrofuran(THF) was purified and dried by distillation from sodium-benzophenone.Trinitrotoluene was supplied courtesy of the Police ScientificDevelopment Branch (PSDB). ¹H NMR analysis: Bruker DPX 400 MHzspectrometer; FTIR analysis: Mattson Galaxy FTIR; Microanalysis:University services; UV-vis Analysis: Perkin Elmer UV-vis spectrometer;SERRS analysis: Renishaw Mark III probe system with excitation bySpectra Physics model 163 air cooled argon ion laser producing an outputof 15 mw at 514.5 nm or a 2020 Renishaw System 1000 microprobespectrometer with excitation by Spectra Physics 2020 water cooled ionargon laser producing a power output at sample of ˜2 mW.

EXAMPLE 1 TNT Detection Using Janowsky Chemistry and Flow Cell Apparatus

[0120] Prior to passing a sample of TNT through the flow cell apparatusas shown in FIG. 2, the TNT must first be captured and transferred tosolution. Vapour from explosive materials present in the atmosphere canbe trapped on “Tenax” (adsorbent polymeric material). Tenax is commonlyused as a trap for Volatile Organic Compounds. Trapping explosive vapouron a solid substrate has the advantage of pre-concentrating the sampleand also enables sampling remote for the detector. When used to captureTNT, the adsorbed material can then be desorbed from the Tenax bywashing with for example acetone.

[0121] The explosive capturing module of the automated TNT detectorconsists of a glass tube containing Tenax supported on glass wool. Airfrom the sampling region is drawn through the glass tube by a fan. Thetube is then placed in the chemistry module of the flow cell system asshown in FIG. 2 where acetone is pumped through it to desorb the TNT.

[0122]FIG. 2 shows schematically the flow cell system used for SERRSdetection of TNT using Janowsky chemistry carried out in situ. Thebinding to colloid and aggregation is also carried out in-line and SERRSspectra detected.

[0123]FIG. 3 shows the SERRS spectrum of the Janowsky complex on colloidpre-aggregated by poly-lysine. The Janowsky complex was formed from a5×10⁻³ M solution of TNT in acetone, to which a few drops of 0.1 M NaOHwere added. 50 μl of this was then added to 1 ml of colloid in a quartzcuvette. The spectrum was recorded in 10 s using 514.5 nm excitation.

[0124] The performance of several different aggregating agents wereinvestigated; poly-lysine nitric acid and sodium nitrate. In FIG. 4 itcan be seen that the best results are obtained with poly-lysine andnitric acid aggregation; salt aggregation gives poor results.Poly-lysine was chosen as the aggregating agent for the Janowsky complexas it gives good SERRS spectra and a reasonable R.S.D. (relativestandard deviation) between successive measurements.

[0125] The amount of poly-L-lysine used as aggregating agent has aprofound effect on the signal intensity measured for the Janowskycomplex.

[0126] The largest SERRS intensity is obtained for the lowest amount ofpoly-L-lysine added. This is due to there being sufficient poly-L-lysinepresent to neutralise the negative charge on the colloid surface, butnot an excess of poly-lysine providing a net positive charge to keep thecolloidal particles apart. This rapidly leads to full aggregation of thesolution and large silver aggregates dropping out of solution; butbefore that happens the enhancement of the SERRS signal is very large.The change in the SERRS intensity over time was also measured and isshown in FIG. 5.

[0127] The R.S.D.'s for change in SERRS intensity with poly-L-lysineaggregation, measured 5 times on one 10⁻³ M. Janowsky complex sample in1 ml colloid are: amount of 0.01% poly-lysine 5 μl 10 μl 20 μl SERRSintensity change 8.67% 1.81% 1.70%

[0128] This shows that although adding a large amount of poly-lysinestabilises the colloid and gives good repeatability, the SERRSintensities are low. Conversely with small amounts of poly-lysine addedthe SERRS intensities are high, but with poorer precision betweensuccessive measurements. Therefore, in the pump system it was decided touse 0.001% poly-lysine solution, which is ten times more dilute thanthat used for the spectra above. The pump has poorer precision comparedto the syringe for dispensing fluids, so even adding large volumes ofthe 0.001% solution to the colloid will only contain a low amount ofpoly-lysine, and will fully aggregate it.

EXAMPLE 2 Reduction of TNT

[0129] a) Reduction of TNT

[0130] The selective reduction of TNT to a specific product isdifficult, however it may be achieved by the following reducing systems(table2). Depending upon the conditions employed a number of reductionproducts may be afforded. TABLE 2 Example of TNT Reducing agentsReducing System Reduction Product(s) Fe/AcOH 2-amino-4,6-dinitrotoluene[5] 4-amino-2,6-dinitrotoluene [6] SnCl₂/HCl/EtOAc2-amino-4,6-dinitrotoluene [5] Indium/NH₄Cl 4-amino-2,6-dinitrotoluene[6] N-(2-Methyl-3,5-dinitro-phenyl)-hydroxylamine [7]N-(3-Methyl-2,4-dinitro-phenyl)-hydroxylamine Borohydride ExchangeResin/MeOH Not characterised Zn/AcOH Not characterised FeSO₄/HCl/MeOH2,4,6-trinitrotoluene FeSO₄/AcOH 2,4,6,-trinitrotoluene

[0131] The selective mono-reduction of TNT was achieved by Fe/AcOH andSnCl₂/HCl respectively. Reduction by Fe/AcOH afforded a mixture of2-amino-4,6-dinitrotoluene and 4-amino-2,6-dintrotoluene in 90% yieldoverall. Separation of both isomers was achieved by crystallisation toyield 29% of the 2-amino derivative. The structure of this isomer wassolved by single crystal X-ray diffraction.

[0132] Reduction by SnCl₂/HCl was shown by proton NMR to be a complexprocess with a number of derivatives formed. Purification of the mixturehowever, afforded the predominant reduction product,N-(2-Methyl-3,5-dinitro-phenyl)-hydroxylamine in 38% yield. Thestructure of this compound was solved by single crystal X-raydiffraction techniques. This method of reduction is completely novel forTNT and demonstrates a useful procedure for the selective reduction of apolynitroaromatic to a dinitro-hydroxylamine.

[0133] To a vigorously stirred solution of TNT (4 g, 17.6 mmol) inglacial acetic acid (88 ml) under argon at room temperature was addedfour portions of iron powder-325 mesh (3.28 g, 58.6 mmol) over twohours, by which time TLC (A) indicated the conversion of startingmaterial. To the solution was added distilled water (80 ml), whichcaused the precipitation of a bright yellow fluffy solid. This solid wascollected by filtration under pressure, washed with copious amounts ofwater and dried at the pump to leave a mixture of [5] and [6] (2.6 g,75%). Recrystallisation of this mixture from ethanol afforded [5] (1 g,29% overall). R_(f) (A) 0.71; ν_(max)/cm⁻¹ 3479, 3387, 1635, 1519, 1341;δ_(H)(400 MHz; Acetone-d₆) 2.30 (3H, s, CH₃) 5.80 (2H, s, NH₂) 7.80 (2H,s, Ar—H); δ_(C)(400 MHz; Acetone-d₆) 13.91 (CH₃) 105.54 (CH) 111.04 (CH)121.89 (C) 147.24 (C) 150.42 (C) 152.50 (C); m/z (EI−HR) 197.04335 [(M)calc. for C₇H₇N₃O₄ 197.04366]; (Found C, 42.32; H, 3.21; N, 21.34.C₇H₇N₃O₄ requires C, 42.64; H, 3.55; N, 21.32%).

[0134] Obtained as per compound [5]. δ_(H)(400 MHz; Acetone-d₆) 2.24(3H, s, CH₃) 5.85 (2H, s, NH₂) 7.33 (2H, s, Ar—H).

[0135] To a stirred solution of TNT (1.4 g, 6.16 mmol) in ethyl acetate(20 ml) was added a solution/suspension of stannous chloride (4.2 g,22.22 mmol) in HCl (7.5 ml). An immediate yellow colour resulted.Stirring was continued until TLC (A) and ninhydrin development indicatedthe conversion of starting materials. The acidic solution was made basicby addition of NaOH solution (1M) and then extracted with saturatedpotassium chloride solution (4×10 ml). The organic layer was dried oversodiumsulphate and purified by column chromatography, eluting withdichloromethane to afford [7] as a yellow/orange solid (500 mg, 38%).Crystallisation from-chloroform afforded [7] as fine orange needles (220mg). R_(f) (B) 0.18; δ_(H)(400 MHz; Acetone-d₆) 2.29 (3H, s, CH₃) 8.04(1H, s, Ar—H) 8.19 (1H, s, Ar—H) 8.39 (1H, s, NOH) 8.42 (1H, s, NOH);m/z (EI−LR) 213 [(M) calc. for C₇H₇N₃O₅ 213]; (Found C, 38.02; H, 2.07;N, 18.84. C₇H₇N₃O₅ requires C, 39.43; H, 3.28; N, 19.72%).

[0136] b) Reduction of TNT Using NaBH₄/Cu(acac)₂/EtOH

[0137] The aim of this experiment was to reduce TNT to a correspondingaromatic amine using copper (II) acetylacetonate and sodium borohydridein ethanol according to a modified version of the method described byHanaya, K. et al. Journal of the Chemical Society, Perkin Transactions,1979, 1, 2409.

[0138] Experimental

[0139] Copper d-acetylacetonate, (0.002 moles, 0.0524 g) in a 50 mlround-bottomed flask was suspended in propan-2-ol (˜2 ml) with stirring.Sodium borohydride (0.001 moles, 0.0378 g) in ethanol was added dropwiseunder nitrogen at room temperature. After heating to 30° C., TNT (0.001moles, 0.227 g), dissolved in propan-2-ol was added, followed by afurther 0.002 moles NaBH (0.0756 g) in ethanol to produce a red colouredcomplex of λ_(max) 497.9 nm. The mixture was stirred at 30° C. for 30mins, then allowed to cool. 5 ml distilled water was added and themixture was extracted with chloroform to give a yellow solution and ared-brown precipitate of the NaBH₄/Cu(acac)₂ complex.

[0140] Analysis

[0141] Thin Layer Chromatography (5:1:1 EtOAc:MeOH:NH₃) showed that allstarting material was converted giving two products (Rf value 0.50 &0.003). The less polar species was identified as oxidisable by iodinecelite development, and gave a red colour by ninhydrin. Infraredspectroscopy indicated the formation of an amine (vNH 3400 & 3490 cm⁻¹)although these bands were not intense.

[0142]¹H NMR (170 MHZ, acetone-d₆, TMS) gave 5.76 ppm (7H, nr s, NH₂)which disappeared on D₂O shake), 8.02 (2H, s, Ar) & 1.29 (?H s, CH₃).However, CHN analysis gave C₇H₁₀N₂ (expect C₇H₇N₃/mono-amino orC₇H₉N₃/di-amino) and a total CHN yield of only 8%.

[0143] c) Reduction of TNT Using NaBH₄/Pd(C)

[0144] The aim of this experiment was to reduce TNT to a correspondingaromatic amine using a palladium catalyst and sodium borohydride,according to a modified version of the route proposed by Petrini et al(Petrini, M.; Ballini, R.; Rosini, G. Synthesis, 1987, 8, 713-714).

[0145] Experimental

[0146] To a 100 ml, 2-necked round-bottomed flask was added TNT (0.001moles, 0227 g) dissolved in TBF (˜4 ml). The mixture was stirred andcooled in an ice-bath. 10% Palladium on activated charcoal (0.04 g) wasadded with stirring, followed by NaBH₄ (0.0031 moles, 0.116 g) and themixture was stirred in ice for 2 hours. After this period, excess NaBH₄was decomposed using HCl (2M) to pH6. Diethyl ether (˜7 ml) was addedfor extraction purposes, and residual solid was filtered off. Thefiltrate was washed twice with 2 ml portions of distilled water, thendried over anhydrous MgSO₄. Solvent was evaporated to give a yellowcoloured, oily product.

[0147] Analysis

[0148] Thin Layer Chromatography (5:1:1 EtOAC:MeOH:NH₃) indicatedcomplete conversion of starting material. Five product spots wereobtained (R_(f) values 0.01, 0.21, 0.32, 0.45 & 0.71) and all wereoxidisable by iodine celite. The spot of R_(f) value 0.32 gave anintense red colour with ninhydrin, indicating the presence of a primaryamine. ¹H NMR confirmed the complex mixture of products; 3 amine signalswere observed (6.22, 6.26 & 6.47 ppm; all disappeared on D₂O shake) and—CH₃ peaks were present at 2.171, 2.209, 2.295 & 2.323 ppm. Aromatichydrogen signals were over a very wide range (7.30-9.13 ppm).

[0149] The selective mono-reduction of TNT was achieved by Fe/AcOH andSnCl₂/HCl respectively. Reduction by Fe/AcOH afforded a mixture of2-amino-4,6-dinitrotoluene and 4-amino-2,6-dintrotoluene in 90% yieldoverall. Separation of both isomers was achieved by crystallisation toyield 29% of the 2-amino derivative. The structure of this isomer wassolved by single crystal X-ray diffraction.

[0150] Reduction by SnCl₂/HCl was shown by proton NMR to be a complexprocess with a number of derivatives formed. Purification of the mixturehowever, afforded the predominant reduction product,N-(2-Methyl-3,5-dinitro-phenyl)-hydroxylamine in 38% yield. Thestructure of this compound was solved by single crystal X-raydiffraction techniques. This method of reduction is completely novel forTNT and demonstrates a useful procedure for the selective reduction of apolynitroaromatic to a dinitro-hydroxylamine.

[0151] The 2-amino-4,6-dintrotoluene derivative prepared by reductionwith Fe/AcOH was successfully diazotised and coupled to form 14 novelazo dyes. The 2-amino-4,6-dinitrotoluene or other 4-amino derivativeobtained from reduction of TNT is diazotised in HCl with sodium nitriteat 0° C. The diazonium salt can then be coupled to any suitable couplingagent, either commercially available or prepared otherwise to give anazo dye. Examples of coupling agents include: Azo Dye(s) Azo Dye(s)Coupling Agent Formed Coupling Agent Formed N,N-dimethyl-1- Yes4-aminobenzotriazole Yes aminonaphthalene 8-hydroxyquinoline Yes1-aminonaphthalene Yes 6-hydroxyquinoline Yes 3,5-dimethoxyphenol Yes5-aminobenzotriazole Yes BONA Yes 3,5-dimethoxyaniline Yes GriessReagent Yes 8-hydroxyquinoline-5- Yes 4-hydroxybenzotriazole Yessulphonic acidDimethyl-[4-(2-methyl-3,5-dinitro-phenylazo)-naphthalen-1-yl]-amine [8]

[0152] To a solution of N,N-dimethyl-1-naphthylamine (0.65 ml, 4 mmol)in sodium acetate buffer (20 ml, pH 6) and acetone (1 ml) was addeddropwise a solution of the diazonium salt of [5] (810 mg, 4 mmol). Afteraddition of the diazonium salt stirring was continued for thirtyminutes. Extraction of the aqueous solution with ethyl acetate (20 ml)and sodium chloride (4×10 ml) afforded a deep purple organic layer,which was dried over sodium sulphate. Purification by columnchromatography, eluting with ethyl acetate (0-5%) in hexane afforded [8]as a purple oil that solidified upon standing. R_(f)(C) 0.26; λ_(max)(MeOH)/nm 505; δ_(H)(400 MHz; DMSO-d₆) 2.91 (3H, s, CH₃) 3.07 (6H, s,N(CH₃)₂) 7.17 (1H, d, J8.6 Nap) 7.64 (1H, t, Nap) 7.76 (1H, t, Nap) 8.02(1H, d, J8.6, Nap) 8.19 (1H, d, J8.3, Nap) 8.60 (1H, s, TNT) 8.77 (1H,s, TNT), 8.93 (1H, d, J8.8, Nap); m/z (FAB) 380.13709 [(M+H)⁺ calc. forC₁₉H₁₈N₅O₆ 380.13588].

[0153] To a solution of 8-hydroxyquinoline (90 mg, 0.68 mmol) in sodiumacetate buffer (20 ml, pH 8) and acetone (1 ml) was added dropwise asolution of the diazonium salt of [5] (135 mg, 0.68 mmol). Afteraddition of the diazonium salt stirring was continued for thirty minutesby which time an orange/brown precipitate had formed. The solids werecollected by filtration and washed with water to afford [9] as a brownsolid (227 mg, 95%). Purification by column chromatography, eluting withmethanol (0-70%) in ethyl acetate (2% ammonia) afforded [9] as a pastyred solid. R_(f) (D) 0.29; λ_(max) (Pyridine)/nm 423, 552; δ_(H)(400MHz; Pyridine-d₅) 2.98 (3H, s, CH₃) 8.29 (1H, d, J8.5, H¹) 8.34 (1H, d,J8.5, H¹) 8.80 (2H, d, J9.3, H^(3,4)) 8.98 (1H, s, NH) 9.04 (2H, s, TNT)9.35 (1H, d, J8.4, H²) 9.39 (1H, d, J8.4, H²); m/z (FAB) 354.08431[(M+H)⁺ calc. for C₁₆H₁₂N₅O₅ 354.08384]. SERRS spectrum shown in FIG. 6.

[0154] To a solution of 5-amino-1H-benzotriazole (68 mg, 0.51 mmol) insodium acetate buffer (50 ml, pH 6) and methanol (20 ml) was addeddropwise a solution of the diazonium salt of [5] (100 mg, 0.51 mmol).After addition of the diazonium salt stirring was continued for thirtyminutes by which time a bright orange precipitate had formed. The solidswere collected by filtration and washed with water and cold methanol toafford [10] as an orange solid (129 mg, 75%). R_(f) (A) 0.54; λ_(max)(MeOH)/nm 363, 478; δ_(H)(400 MHz; DMSO-d₆ {D₂O shake}) 2.43 (3H, s,CH₃) 6.87 (1H, d, J9.1, BT) 7.83 (1H, d, J9.0, BT) 8.07 (2H, s, NH₂,disappeared) 8.40 (2H, s, TNT) 13.37 (1H, s, NH, disappeared) and 2.70(3H, s, CH₃) 7.50 (1H, d, BT) 7.91 (1H, d, J8.8, BT) 8.07 (2H, s, NH₂,disappeared) 8.53 (1H, s, TNT) 8.57 (1H, s, TNT) 15.36 (1H, s, NH,disappeared); m/z (EI−HR) 342.08310 [(M) calc. for C₁₃H₁₀N₈O₄342.08250]. SERRS spectrum shown in FIG. 7.

[0155] In order to exploit the strong surface complexing capabilities ofbenzotriazole several novel coupling compounds incorporating abenzotriazole linker were prepared. These compounds were designed tofacilitate both optimal surface interactions and efficient coupling ofthe TNT diazonium cation. The coupling agents were prepared by reductiveamination of 5-aminobenzotriazole with a suitable aldehyde.

[0156] General Reductive Amination Reaction

[0157] Only aldehydes containing an electron rich phenyl ring ornaphthalene ring system were employed (Table X) The aldehydes werechosen in order to promote rapid and efficient coupling with the TNTdiazonium cation, which is a relatively poor electrophile. Aldehydesemployed in reductive amination with 5-aminobenzotriazole Yield of Amineafter Aldehyde Purification Naphthalene-1-carbaldehyde 64%Naphthalene-2-carbaldehyde 80% 3,5-dimethoxybenzaldehyde 60%

[0158] The aldehydes all underwent smooth reductive amination with5-aminobenzotriazole. Purification by column chromatography was used toafford moderate to high yields of the corresponding amines.

[0159] Azo coupling of each amine with diazotised [5] occurred rapidlyin a MeOH/NaOAc buffer to precipitate the azo dyes as bright red solids.Crystallisation of the 2-naphthylamine-azo [14] derivative from acetoneresulted in the formation of thin red needles. The structure of this dyewas solved by single crystal X-ray diffraction to provide the firstexample of an azo dye incorporating the structure of TNT. Furthermore,X-ray analysis was also able to reveal that coupling of the diazoniumsalt had occurred on the benzotriazole ring and not the naphthalene ringas first expected.

[0160] Comparison of the proton NMR of both naphthalene azo dyes showedthat in each case the coupling positions were consistent on thebenzotriazole ring. In the case of the dimethoxybenzene coupling agent,the proton NMR clearly demonstrated that attack of the diazonium cationalso occurred on the benzotriazole ring.

[0161] Each of the dyes exhibited a strong absorption in the visiblecoincidental with the frequency of SERRS excitation at 514 nm (see tablebelow) and therefore SERRS analysis of the dyes was undertaken at thiswavelength. SERRS spectrum of dyes 14 and 15 shown in FIG. 8a. UV-visabsorption maxima of benzotriazole-TNT-azo dyes Dye λ_(max) (DMF)/nm[14] 504 [15] 362, 507 [16] 362, 506(1H-Benzotriazol-5-yl)-naphthalen-2-ylmethyl-amine [11]

[0162] The general procedure employed was as follows.Naphthalene-2-carbaldehyde (624 mg, 4 mmol) in methanol (25 ml) wasadded to a solution of 5-amino-1H-benzotriazole in methanol (25 ml) thathad been adjusted to pH 6 by the addition of glacial acetic acid (1 ml).Sodium cyanoborohydride (1.89 g, 30 mmol) was added and the resultingmixture was stirred at room temperature overnight by which time TLC (A)and ninhydrin development indicated the conversion of startingmaterials. Hydrochloric acid (50% HCl/H₂O, 5 ml) was added and thesolution was neutralised to pH 7 by addition of NaOH (1M). Methanol wasremoved in vacuo to afford an aqueous residue that was dissolved inethyl acetate (50 ml) and extracted with sodium chloride solution (4×20ml). Purification by column chromatography, eluting with methanol(0-10%) in dichloromethane afforded [11] as a pale yellow oil.Trituration from diethyl ether afforded [11] as a white solid (880 mg,80%). R_(f) (A) 0.42 δ_(H)(400 MHz; DMSO-d₆ {D₂O shake}) 4.50 (2H, s,CH₂) 6.45 (1H, s, BT) 6.89 (1H, s, NH, disappeared) 6.91 (1H, d, J8.9,BT) 7.43-7.49 (2H, m, Ar—H) 7.54 (1H, d, J8.6, Nap) 7.65 (1H, d, J8.9,BT) 7.82-7.89 (4H, m, Nap) 14.72 (1H, s, NH, disappeared); m/z (EI−HR)274.12177 [(M calc. for C₁₇H₁₄N₄ 274.12185].

[0163] As per the general procedure for compound [11]. Trituration fromdiethyl ether afforded [12] as a white solid (700 mg, 64%). R_(f) (A)0.30 δ_(H)(400 MHz; DMSO-d₆ {D₂O shake}) 4.75 (2H, s, CH₂) 6.49 (1H, s,BT) 6.77 (1H, s, NH, disappeared) 6.91 (1H, d, J9.0, BT) 7.43 (1H, t,Nap) 7.51-7.59 (3H, m, Nap) 7.65 (1H, d, J9.0, BT) 7.82 (1H, d, J8.1,Nap) 7.94 (1H, d, J8.4, Nap) 8.14 (1H, d, J8.1, Nap) 14.74 (1H, s, NH,disappeared); m/z (EI−HR) 274.12148 [(M) calc. for C₁₇H₁₄N₄ 274.12185].

[0164] As per the general procedure for compound [11]. Trituration fromdiethyl ether afforded [13] as a white solid (685 mg, 60%). R_(f) (A)0.29 δ_(H)(400 MHz; DMSO-d₆ {D₂O shake}) 3.69 (6H, s, 2×OMe) 4.25 (2H,s, CH₂) 6.34 (1H, s, DMB) 6.39 (1H, s, BT) 6.54 (2H, s, DMB) 6.73 (1H,s, NH, disappeared) 6.85 (1H, d, J8.9, BT) 7.64 (1H, d, J8.9, BT) 14.76(1H, s, NH, disappeared); m/z (EI−HR) 284.12718 [(M) calc. forC₁₅H₁₆N₄O₂ 284.12733].

[0165] The general procedure employed was as followed. To a solution ofcompound [11] (274 mg, 1 mmol) in sodium acetate buffer (20 ml, pH 6)and methanol (40 ml) was added dropwise a solution of the diazonium saltof [5] (197 mg, 1 mmol). After addition of the diazonium salt stirringwas continued for thirty minutes by which time a bright red precipitatehad formed. The solids were collected by filtration and washed withwater and cold methanol to afford [14] as a red solid (275 mg, 57%).R_(f) (A) 0.79; λ_(max) (DMF)/nm 504; δ_(H)(400 MHz; DMSO-d₆) 4.94 (2H,s, CH₂) 7.10 (11H, d, J9.3, BT) 7.44-7.51 (2H, m) 7.59 (1H, d, J8.4,Nap) 7.82-7.94 (4H, m, Ar) 8.01 (1H, d, J11.2, Nap) 8.65 (1H, s, TNT)8.79 (1H, s, TNT) 9.88 (1H, s, NH) 15.67 (1H, s, NH); m/z (FAB)483.15553 [(M+H)⁺ calc. for C₂₄H₁₉N₈O₄ 483.15293]. X-ray structure isshown in FIG. 8b.

[0166] As per the general procedure for compound [14] to afford [15] asa red solid (400 mg, 81%). R_(f) (A) 0.72; λ_(max) (DMF)/nm 362, 507;δ_(H)(400 MHz; 1)MS0-d₆) 2.59 (3H, s, CH₃) 3.71 (6H, s, 2×OMe) 4.68 (2H,s, CH₂) 6.43 (1H, s, DMB) 6.60 (2H, s, DMB) 7.04 (1H, d, J8.9, BT) 8.05(1H, d, J9.1, BT) 8.67 (1H, s, TNT) 8.77 (1H, s, TNT) 9.75 (1H, s, NH)15.68 (1H, s, NH); m/z (FAB) 494.16970 [(M+H)⁺ calc. for C₂₂H₂₂N₈O₆494.16623].

[7-(2-Methyl-3,5-dinitro-phenylazo)-1H-benzotriazol-6-yl]naphthalen-1-ylmethyl-amine[16]

[0167]

[0168] As per the general procedure for compound [14] to afford [16] asa red solid (250 mg, 52%). R_(f) (A) 0.68; λ_(max) (DMF)/nm 362, 506;δ_(H)(400 MHz; DMSO-d₆) 1.98 (3H, s, CH₃) 5.24 (2H, s, CH₂) 7.19 (1H, d,J9.2, BT) 7.48-7.62 (4H, m, Ar) 7.94 (1H, d, J8.1, Nap) 8.00 (1H, t,Nap) 8.12 (1H, d, J9.3, BT) 8.14 (1H, t, Nap) 8.58 (1H, s, TNT) 8.79(1H, s, TNT) 10.00 (1H, s, NH) 15.85 (1H, s, NH); m/z (FAB) 483.15179[(M+H)⁺ calc. for C₂₄H₁₉N₈O₄ 483.15293].

[0169] e) Preparation of an Imine from a Pure TNT Derivative

[0170] The aim of the experiment was to produce a Schiff's Base from apure amine-derivative of TNT, which is commercially available. The aminechosen was 2,4,6-Triaminotoluene trihydrochloride (TAT), atri-aminoderivative.

[0171] Experimental

[0172] Tri-aminotoluene trihydrochloride (0.001 moles) was dissolved inthe minimum volume of methanol. The aldehyde coupling agent,1-hydroxynaphthaldehyde, (1 mol.equiv.) was also dissolved in theminimum volume of methanol and added drop-wise with stirring to thesolution of the amine. The mixture was allowed to stir for 1 h, afterwhich time solvent could be filtered off to give the orange-yellowcoloured imine in good yield.

[0173] Analysis

[0174] Thin Layer Chromatography (9:1 DCM:MeOH) showed that all the TAThad been coverted to 3 products, one of which was yellow coloured.UV-visible absorption spectrometry (EtOH) showed that the compoundformed absorbed at 456 & 367 nm. FTIR showed vC=N at 1618 cm⁻¹,confirming imine formation, and a very broad vOH from 3400-3600 cm⁻¹which probably masked vNH. Mass Spectra (FAB⁺) showed that the mono-,di- and tri-substituted imine derivatives of TAT, (m/z) 292, 446 & 601respectively were all present. SERRS analysis of the self-aggregatedmixture (50 μl of ˜10⁻⁵M imine in 2 ml colloid) showed a fairly simpleand characteristic spectrum with important peaks at 1349, 1401, 1452 &1605 cm⁻¹ as shown in FIG. 9.

EXAMPLE 3 Synthesis of 5-Carboxaldehyde-1H-Benzotriazole

[0175] a) Synthesis of 1H-Benzotriazole-5-Diazonium Chloride

[0176] 4-amino-1H-benzotriazole (5 g, 37.5 mmol) was dissolved in theminimum volume of 50% HCl/H₂O(10 ml) and cooled on an ice-bath to 0° C.A solution of NaNO₂ (2.75 g, 41 mmol) in distilled water (5 ml) wasadded dropwise to the amine solution with vigorous stirring. Aftercomplete addition, the resulting solution was stirred for 15 minutes at0° C., then made neutral to pH7 by addition of aqueous sodium acetatesolution (1M).

[0177] b) Synthesis of 5-Carboxaldehyde-1H-Benzotriazole

[0178] Formaldoxime (5 g, 37.5 mmol) was weighed out and made up to 50ml in distilled water to yield a 10%. aqueous solution. To this wasadded copper sulphate (925 mg, 3.75 mmol), sodium sulphite (150 mg, 1.10mmol) and 4 g of sodium acetate in 10 ml of water. The solution wasmaintained at 10-15° C. by means of a cold water bath and stirredvigorously. The neutral diazonium salt prepared above in a) was thenslowly introduced below the surface of the formaldoxime solution bysiphoning under slight nitrogen pressure. After addition of thediazonium salt was complete, the mixture was stirred for an additionalhour. To this stirred solution was added 50% HCl/H₂O (50 ml), followedby gentle refluxing overnight under an atmosphere of nitrogen. TLC usingdichloromethane (DCM):MeOH (9:1) as a solvent treatment with2,4-dinitro-phenylhydrazine revealed the presence of an orange spot,indicative of the aldehyde. The remaining solution and tan precipitatewere neutralised by addition of sodium bicarbonate to pH 7. Removal ofwater in vacuo left a tan coloured solid, which was purified by columnchromatography on silica using a DCM-MeOH gradient (0-10%). Triturationof the combined pure fractions with diethyl ether yielded a pale yellowsolid (1.1 g, 20%). R_(f) DCM:MeOH (9:1) 0.39; (Found C, 57.02; H 3.14;N, 30.87. C₇H₅N₃O requires C, 57.14; H, 3.40; N, 28.57%); v_(max)(KBr)/cm⁻¹ 3175, 2918, 2866, 2753, 1639, 1312, 1211; δ_(H)(400 MHz;CD₃OD) 7.94 (1H, d, J 8.0, Ar—H), 8.02 (1H, d, J 8.7, Ar—H), 8.53 (1H,s, Ar—H), 10.13 (1, s, CHO): m/z (El) 147.

[0179] The most suitable method for the synthesis of5-carboxyaldehyde-1H-benzotriazole was a route previously described byBeech (Beech, W. F. Journal of the Chemical Society 1954, 1297) andothers (Manecke, G.; Ehrenthal, E.; Finck, W.; Wunsch, F. Israel Journalof Chemistry 1978, 17, 257-263) for the preparation of aromaticaldehydes and ketones from diazonium salts. By diazotisation of anaromatic primary amine and reaction with formaldoxime, followed byhydrolysis, aromatic aldehydes were obtained in moderate yields. Thismethod is particularly attractive for benzotriazole for several reasons.Most importantly, 5-amino-1H-benzotriazole is a commercially availablematerial, which is known to form diazonium salts with ease. Therefore,the synthesis of starting material from benzotriazole itself was notrequired. Furthermore, the chemistry involved was simple and comprisedof only one step. Protection of functional groups was not required andtherefore reaction times were minimised, hopefully resulting in anoverall increase in the yield of aldehyde over other more complexmethodologies.

[0180] Thus, a novel method for the preparation of5-carboxyaldehyde-1H-benzotriazole based upon a modified version of themethod described by Beech was achieved. The synthetic route employed wasrelatively simple, however a 20% yield of aldehyde was obtained.

[0181] The SERS spectrum observed for the aldehyde was strong andcharacteristic (FIG. 10). The presence of a strong carbonyl band at 1600cm⁻¹ was not immediately apparent, although a shoulder on the aromaticstretch at 1600 cm⁻¹ may be due to carbonyl vibrations.

EXAMPLE 4 Synthesis of 1H-Benzotriazole-2,4-Dinitrophenylhydrazone

[0182]

[0183] The reaction of 5-carboxaldehyde-1H-benzotriazole with2,4-dinitrophenylhydrazine to form a simple hydrazone is a chemical testto prove the presence of the aldehyde was attempted to demonstratereactivity.

[0184] A solution of 2,4-dinitrophenylhydrazine (19.3 mg, 0.1 mmol) and5-carboxyaldehyde-1H-benzotriazole (14.7 mg, 0.1 mmol) in MeOH (10 ml)with three drops of 50% Hcl/H₂O was refluxed gently overnight to yieldan orange precipitate. The precipitate was filtered and washed with coldMeOH to give an orange solid (15 mg, 55%). v_(max) (KBr)/cm⁻¹ 3098,3010, 2356, 1774, 1609, 1589, 1512; δ_(H)(400 MHZ; d₆-dmso) 4.17 (1H, brs, N—H), 7.98 (1H, d, J 8.7, Ar—H), 8.02 (1H, d, J 8.7, Ar—H), 8.16 (1H,d, J 9.6, Ar—H), 8.23 (1H, s, CH═N), 8.40 (1H, d, J 9.5, Ar—H), 8.86(2H, s, Ar—H), 11.73 (1H, s, N—H); m/z (El) 327.

[0185] The orange solid of 1H-benzotriazole-2,4-dinitrophenylhydrozoneproduced a characteristic and strong SERRS spectrum (see FIG. 11). Thisdemonstrated that the aldehyde was reactive to the same functionality asthe expected reaction product of RDX.

EXAMPLE 5 Derivatisation of TNT and Detection by SERRS

[0186] Synthesis of 2,4,6-trinitrostyryl-5-methylthiophene

[0187] The general procedure used was as follows. A solution of TNT (50mg, 0.22 mmol) and 5-methyl-thiophenecarboxaldehyde (0.025 ml, 0.22mmol) in dry THF (5 ml) was gently refluxed for three hours with twodrops of piperidine; by which time thin layer chromatography [DCM:MeOH(9:1)] showed complete conversion of starting materials. THF was removedin vacuo to leave a dark red oily residue, which was dissolved in ethylacetate (20 ml) and extracted with potassium chloride solution (4×20ml). The organic layer was dried and purified by column chromatographyon silica, eluting with a DCM-MeOH gradient (0-10%), to yield a redsolid. Recrystallisation from ethanol gave [1] as small red needles (30mg, 41%); R_(f) [DCM:MeOH (9:1)] 0.79; mp 160-161° C. (lit., (Buu-Hoi N.P.; Hoan, N.; Lavit, D. Journal of the Chemical Society 1950, 2130)164-165° C.); (Found C, 46.60; K, 2.42; N, 12.04. C₁₂H₉N₃O₆S requires C,46.43; H, 2.68; N, 12.50%); λ_(max)(MeCN)/nm 408; v_(max)(KBr)cm⁻¹ 3101,3063, 1617, 1593, 1532, 1443, δ_(H)(400 MHz; CDCl₃) 2.53 (3H, s, CH₃),6.72 (1H, d, J 3.6, Ar—H), 6.88 (1H, d, J 16.2, vinyl-H), 6.98 (1H, d, J16.2, vinyl-H), 6.98 (1H, d, J 3.6, Ar—H), 8.79 (2H, s Ar—H).

[0188] Synthesis of 2,4,6-trinitrostyryl-1H-benzotriazole

[0189] As per the general procedure described above with the exceptionthat 0.1 mmol of TNT and 1H-benzotriazole-5-carboxyaldehyde wereemployed.

[0190] The aim was to produce coloured SERRS active TNT derivatives, bycondensation with aromatic aldehydes.

[0191] The condensation of TNT with 5-methyl-thiophenecarboxaldehyde toafford 2,4,6-trinitrostyryl-5-methylthiophene was demonstrated as aneffective method for the derivatisation and detection of TNT by SERRS,producing an intense and characteristic spectrum at low concentration(see FIG. 12).

[0192] The synthesis of 2,4,6-trinotrostyryl-5-methlthiophene was asignificant result, demonstrating for the first time that themolecularly specific detection of TNT by SERRS can be achieved at ultralow concentrations using simple chemistry requiring very little samplepreparation. This was in contrast to the previous derivatisationtechniques, such as reduction and complex formation which were eithertime consuming or not sensitive enough.

[0193] Extensive research has shown that coloured benzotriazolederivatives are capable of producing strong and characteristic SERRSspectra. Benzotriazole has been shown to bond irreversibly to variousSERRS substrates including silver, in an orientation almostperpendicular to the metal surface. As such, it was decided toincorporate benzotriazole in the TNT condensation derivative utilising abenzotriazole aldehyde derivative (1H-benzotriazole-5-carboxaldehyde).

[0194] The unpurified reaction mixture of2,4,6-trinitrostyryl-1H-benzotriazole displayed a strong andcharacteristic SERRS spectrum (see FIG. 13) markedly different to thespectrum observed from 2,4,6-trinitrostyryl-5-methylthiophene. Byincorporation of benzotriazole into the TNT derivative, a novel colouredmolecule primed to experience maximum surface enhancement was produced.A strong SERRS spectrum was obtained prior to any purification andwithout the use of aggregation by poly-L-lysine.

[0195] Therefore, by utilising a benzotriazole aldehyde the sensitivedetection of TNT by SERRS is now possible. The derivation involvedrequires a simple one step reaction, simpler than the previouslyemployed two stage reduction chemistry. Furthermore, complex mixturesare not a consideration as the functionality under exploitation is thesingle TNT methyl group and not the nitro groups. Therefore, the needfor purification prior to the colour formation step is removed. Theresult of this to save analysis time considerably and to increasedetection limits as none of the TNT derivative will be lost, which is acertainty if the reduction chemistry were employed. The incorporation ofbenzotriazole into the TNT derivative ensures that detection limits andreproducibility are sure to be improved significantly, as is the speedand simplicity of the technique. In fact, the only purification stepnecessary with this method of derivatisation may be the removal orunreacted aldehyde, which may interfere by producing its own SERSspectrum. This may not be a serious problem however, as the limit ofdetection of the aldehyde is approximately 10⁻⁵M. Therefore, it islikely that any SERRS spectrum from a coloured TNT-benzotriazolecompound would be observed over the SERS spectrum of excess aldehyde.

EXAMPLE 6 Preparation of Suitable Schiff Bases

[0196] A number of suitable Schiff bases were synthesised. These wereall prepared by the same general procedure.

[0197] Equimolar amounts (0.005 moles) of each aldehyde and amine wereweighed. The amine was placed in a 250 ml round bottomed flask anddissolved in methanol. The aldehyde was placed in a 100 ml conical flaskand dissolved in methanol. Under constant stirring, the aldedhyde wasadded drop-wise into the amine solution over a period of 20 minutes.This solution was then left stirring overnight. The product was vacuumfiltered and dried over phosphorus pentoxide in a vacuum dessicator.Thin Layer Cromatography Systems (TLC) System (A)Dichloromethane/methanol 9:1 ratio System (B) Dichloromethane/methanol8:2 ratio System (C) Ethyl acetate/ammonia/methanol 5:1:1 ratio

[0198] To determine the purity of the Schiff bases TLC were obtained anda ninhydrin test was carried out to determine if a primary amine waspresent. The Schiff bases containing an excess of primary amine werepurified by recrystallisation.

[0199] A ninhydrin solution was used for the detection of primary amineson the TLC plates. This was approximately a 1% w/w solution ofninhydrine in ethanol.

[0200] Schiff Base(9)

[0201] 2-(napthalen-2-ol)-methyleneamine-benzoic acid

[0202] 0.8608 g of 2-hydroxyl-1-napthaldehyde was dissolved in 35 mlmethanol and added to a solution of 0.6855 g anthranilic acid (o-aminebenzoic acid) dissolved in 10 ml methanol. A yellow compound wasobtained.

[0203] λ_(max)=440 nm.

[0204] δ_(H)(400 MHz; DMSO-d₆); 7.8 (1H, d, aromatic H): 7.2 (1H, m,aromatic H); 7.3 (1H, m, aromatic H); 7.5 (1H, m, aromatic H); 7.7 (2H,dd, aromatic H); 7.8 (1H, d, aromatic H); 7.9 (2H, t, aromatic H); 8.3(1H, d, aromatic H); 9.3 (1H, s, C—H). R_((f))(C)=0.66.

[0205] Schiff Base(11)

[0206] (1′H-benzotriazole-5′-yl)-1-iminomethyl-napthalen-2-ol

[0207] 0.4308 g of 2-hydroxy-1-napthaldehyde was dissolved in 18 mlmethanol and added to a solution of 0.3353 g 5-aminobenzotriazoledissolved in 16 ml methanol (0025 mol. amounts were used). Ayellow/orange compound was obtained.

[0208] (Found: C, 68.9; H, 2.9; n, 18.4; C₁₇H₁₂N₄O. Requires: C, 70.8;H, 4.2, N, 19.4%0.

[0209] λ_(max)=445 nm.

[0210] δ_(H)(400 MHz; DMSO-d₆); 4.1 (1H, s, OH); 7.1 (1H, d, aromaticH); 7.3 (1H, t, aromatic H); 7.5 (1H, t, aromatic H); 7.6 (1H, d,aromatic H); 7.8 (1H, d, aromatic H); 7.9 (1H, d, aromatic H); 8.2 (1H,s, aromatic H); 8.5 (1H, d, aromatic H); 9.8 (1H, s, c-H).

[0211] R_((f))(C)=0.59.

[0212] Schiff Base(13)

[0213] (1′H-benzotriazole-5′-yl)-4-iminomethyl-benzyl-1,3-diol

[0214] 0.1676 g of 2,4-dihydroxybenzaldehyde was dissolved in 10 mlmethanol and added to a solution of 0.1725 g 5-aminobenzotriazoledissolved in 16 ml methanol (0.00125 mol. amounts were used). A yellowcompound was obtained.

[0215] (Found: C, 61.3; H, 4.0; N, 21.8; C₁₃H₁₀N₄O₂. Requires: C, 61.4;H, 4.0, N, 22.0%).

[0216] λ_(max)=345 nm

[0217] δ_(H)(400 MHz; DMSO-d₆); 6.3 (1H, s, aromatic H); 6.4 (1H, d,aromatic H); 7.4 (2H, d, aromatic H); 7.7 (1H, s, aromatic H); 7.9 (1H,d, aromatic H); 8.9 (1H, s, aromatic H).

[0218] R_((f))(C)=0.83.

[0219] Colloid Preparation

[0220] The silver colloid was prepared by using a modified Lee andMeisel (P. C. Lee & D. Meisel, J. Phys. Chem., 1982, 86, p3391)procedure. All glassware was thoroughly cleaned by soaking overnight inaqua regia (HCl: HNO₃, 4:1 v/v) and then washed with a soap solution andrinsed well with distilled water. 500 ml of distilled water was placedin a 1 litre round bottomed flask, heated to approximately 45° C. andwhile constantly stirring 90 mg of silver nitrate was added. Thissolution was heated to almost boiling and 10 ml of a 1% solution oftri-sodium citrate was added. The heat was reduced and the solution waskept gently boiling (at 98° C.) with constant stirring. Once thesolution had cooled, its quality was assessed using U.V-Visiblespectroscopy. A small volume of the colloid was diluted with distilledwater and run along with a blank. The colloid should preferably have anabsorption maximum of 404 nm±2 nm, with a full width half height of thispeak less than 60 nm.

[0221] The colloid used for analysis had a λ_(max) of 408 nm with a fullhalf width of 80 nm.

[0222] Preparation of Samples for SERRS Analysis

[0223] The samples were prepared in two different ways.

[0224] The first method: 500 μl of distilled water was added to 500 μlof colloid, 100 μl of a Schiff base solution was added to this.

[0225] The second method: 500 μl of distilled water was added to 500 μlcolloid and 100 μl of a 0.01% Poly-L-lysine solution was added foraggregation. 100 μl of a Schiff base solution was also added.

[0226] In-situ Analysis

[0227] In-situ analysis was undertaken for Schiff base (11) using the514.5 nm probe. This was done following two different procedures.

[0228] A flow cell was used for the first procedure. Colloid and5-aminobenzotriazole were pumped through first,2-hydroxyl-1-napthaldehyde was then pumped into the flow cell. Aspectrum was obtained two and four minutes after the pump was started.The pump speed was reduced to 10 rpm and after two minutes a spectrumwas obtained.

[0229] For the second procedure the colloid was re-spun in methanol.Approximately 5 ml of very concentrated colloid was placed in a vial. Tothis a small spatula full of 5-aminobenzotriazole was added (˜1-2 mg),this was left for approximately 1 hr to allow the benzotriazole group tobind onto the metal surface. A small spatula full of2-hydroxy-1-napthaldehyde (˜1-2 mg) was added to the colloid solution.This was left to react over a weekend. A spectrum of the solution wasthen obtained.

[0230] To this solution a small spatula full of zinc acetate was added(1-2 mg). A precipitate formed immediately, however the solution wasleft overnight to allow the zinc acetate to react completely. Therelevant information was obtained from this solution using the 514.5 nmprobe.

EXAMPLE 7 Use of Benzotriazole Maleimide to Provide Hemoglobin SERRS

[0231] When hemoglobin is added to silver colloid and aggregated thereare no signals obtained corresponding to the heme moiety, even whenincubated overnight with poly(L-lysine) see FIG. 14. This is due to theinability of the hemoglobin to adsorb efficiently to the colloidalsurface. In order to overcome this adsorption problem a reactivebenzotriazole moiety was synthesised to react with functional groups onthe protein and then pull the protein onto the metal surface. In thisexample benzotriazole maleimide was synthesised which in its own rightis SERS but not SERRS active. The benzotriazole functionality complexesto the surface and the maleimide group reacts with any thiol groupspresent in the protein. In this case hemoglobin from horse has twostrands with 2 cysteines available for derivatisation in the protein(Human hemoglobin has 4 strands with 6 cysteines available forderivatisation). Thus by reacting the cysteine residues available withthe benzotriazole maleimide the surface adsorption part of the SERRS isprovided by the benzotriazole and the resonance effect from the hememoiety.

[0232] By derivatising the hemoglobin with benzotriazole SERRS signalswere obtained that were specific to the heme moiety thus demonstratingthe need for efficient surface adsorption to provide strong SERRS from aspecies that does not normally provide SERRS see FIG. 15. Similarlyother functional groups could be added to benzotriazole to act in asimilar fashion. For example a benzotriazole succinimide could be usedto couple to free amine groups in the protein such as the N-terminus oravailable lysine residues.

[0233] Synthesis of 3-[Benzotriazo-5′-yl]carbonyl-acrylic acid

[0234] Maleic anhydride (1.1816 g, 0.012 moles) was dissolved indichloromethane (30 ml) and 5-aminobenzotriazole (0.9073 g, 0.007 moles)dissolved in acetone (5 ml) added dropwise. The resulting suspension wasstirred at room temperature for 4 hours by which time TLC, (ethylacetate/methanol/ammonia, 5:1:1), showed complete reaction. The productwas isolated by filtration, washed with acetone and dried to produce thetitle compound in 97% yield, δ_(H) [(CD₃)₂)SO] 6.32-6.35 (1H, d, J 12.0HC═CH) 6.51-6.54 (1H, d, J 12.0 HC═CH) 7.37 (1H, d, ar, BT) 7.96 (1H, d,ar, BT) 7.96 (1H, s, ar, BT) 10.65 (1H, s, triazole) 13.0 (1H, s, NHCO)15.53 (1H, s, COOH).

[0235] Synthesis of 1-[1′-acetyl-benzotriazo-5′-yl]-pyrroloe-2,5-dione

[0236] Anhydrous sodium acetate (0.2606 g, 0.003 moles) was dissolved inacetic anhydride (25 ml) and compound (0.487 g, 0.002 moles) addedslowly. The resulting suspension was refluxed at 90° C. for 30 minutesby which time TLC, (dichloromethane/methanol, 9:1), showed completereaction. After removal of the acetic anhydride in vacuo, the productwas collected by filtration, washed with ice cold water, petroleum ether(bp 40-60° C.) and dried. Analysis of the product showed a mixture ofproducts. The main product being the acetylated benzotriazole maleimidewith the desired product as a minor product.

[0237] Synthesis of 1-Benzotriazo-5′-yl-pyrrole-2,3-dione

[0238] The mixture from the previous step (0.47 g) was dissolved intritluoroacetic acid (10 ml) and the reaction was followed by TLC,(ethyl acetate/hexane, 8:2). After 3 days, TLC showed reaction wascomplete. Trifluoroacetic acid was removed in vacuo to give a brown oilwhich upon co-evaporation with methanol left a yellow solid, which wascollected by filtration and washed with ice cold water and petroleumether (bp 50-60° C.) to yield the title compound in 60%, δ_(H)[(CD₃)₂CO] 7.08 (2H, s, maleimide) 7.8-8.2 (2H, s, ar, BT) 14.8 (1H, s,triazole), λ_((max))=278.9 nm.

[0239] Coupling of Benzotriazole Maleimide to Horse Hiemoglobin

[0240] Horse hemoglobin (0.0955 g, 1.481 μmol) was dissolved in TSMZbuffer (25 ml) and benzotriazole maleimide (0.0101 g, 31 μmol) dissolvedin DMF (1 ml) added. The resulting solution was stirred at roomtemperature for 6 hours then stored in the fridge overnight. Gelfiltration through a PD-10 column (Sephadex G-25) using TSMZ buffer asthe eluant removed any unreacted benzotriazole maleimide.

EXAMPLE 8 RDX Reduction and SERRS Detection

[0241] (1) Hydrazine Formation—Pyridine Azine

[0242] Sodium metal (400 mg, 16.65 mmol) was dissolved in mercury (9.6g, 42.65 mmol) to yield a 4% amalgam. RDX (222 mg, 1.0 mmol), dissolvedin dry THF-(10 ml) was poured onto the amalgram under argon and themixture was stirred. Water (5 ml) was added dropwise over 5 minutes, bywhich time TLC (dichloro methane-nethanol 9:1) indicated the conversionof RDX to a single product (Rf 0.0), which produced a positive ninhydrinreaction.

[0243] The basic solution was neutralised to pH 7 with acetic acid (10ml). Pyridine-4-carboxaldehyde (0.3 ml, 3 mmol) was added dropwise tothis solution with stirring, resulting in the formation of a strongyellow colour. After 5 minutes a yellow crystalline solid hadprecipitated. The solid was filtered and washed with cold water to yieldsmall yellow crystals. ¹H NMR (400 MHz, CDCl₃) δ 7.68 (4H, d, Ar—H),8.55 (2H, s, imine), 8.75 (4H, d, Ar—H); Found C, 67.79; H, 3.59; N,26.11. C₁₂H₁₀N₄ requires C, 68.57; H, 4.76; N, 26.66%; m/z EI 210.091(78.48%).

[0244]FIG. 16 shows the SERRS spectrum of the hydrazine compound.

[0245] (2)(i) Hexamine Formation

[0246] To a solution of Ni(OAc)₂ (38 mg, 0.15 mmol) in dry methanol (50ml) was added BER (2 g, 4.5 mmol). After stirring for one minute RDX(111 mg, 0.5 mmol) was added and stirring was continued at roomtemperature for thirty minutes. BER (2 g, 4.5 mmol) was added again tothe reaction mixture and stirring was continued. The solution was leftto stir overnight, by which time TLC (dichloromethane-methanol 9:1)indicated conversion of RDX to a product (R_(f) 0.0), which produced apositive ninhydrin reaction. BER was removed by filtration and solventwas removed to yield an oily residue. Dissolution of the residue in D₂Oand subsequent ¹H NMR revealed the presence of a single broad peak at4.75 ppm, consistent with an authentic sample of hexamine.

[0247] (ii) ¹H NMR Study of BER-Ni(OAc)₂ Reduction

[0248] Reduction was carried out according to the procedure described insection 2 (i), with the exception that deuterated methanol (d₄-methanol)was used as the reaction solvent. In order to ensure sample homogeneity,each aliquot of solution was filtered through cotton wool prior toanalysis. A ¹H NMR spectrum of the reaction mixture was taken every tenminutes for a period of one hour followed by two spectra after two andthree hours, which confirmed the complete consumption of RDX. NMRspectra were acquired for reduction mixture aliquots at the reactiontime intervals quoted. The initial measurement, at zero minutes wastaken of an aliquot of the reaction mixture sampled immediately afterRDX was added to the BER solution. Further measurements were then madeat the time intervals as indicated on the graph. The results shown thatRDX is consumed to yield a new product, which appears at 4.8 ppm. Thisis consistent with hexamethylenetetramine, which appears at 4.76 ppm inD₂O (see FIG. 17).

[0249] (iii) Hexamine Azo Dye

[0250] 5-amino-benzotriazole (134 mg, 1 mmol) was dissolved in 50% Hcl(2 ml) and diazotised by dropwise of NaNO₂ (104 mg, 1.5 mmol) at 0° C.The diazonium salt formed was added dropwise to a solution of hexamine(70 mg, 0.5 mmol) in sodium acetate buffer (5 ml. The solution wasstirred for 5 mins and neutralised to pH 7 by addition of saturatedsodium carbonate solution. The resulting aqueous solution was extractedwith ethyl acetate (3×10 ml) and saturated NaCl solution (3×10 ml) andthe organic layer was dried over sodium sulphate. Purification by columnchromatography (dichloromethane:methanol, 0-10%) yielded the product asa red powder. M/z 420.4 (M+2).

[0251] The SERRS spectrum of the hexamine azo dye is shown in FIG. 18.

EXAMPLE 9 I) Novel Modified Janowsky Chemistry

[0252] The formation of a Janowsky complex is a classical test for thepresence of a nitroaromatic or a ketone.^((1,86)) Deprotonation of theketone to yield an enolate anion and subsequent attack upon the electrondeficient nitroaromatic leads to the formation of a coloured σ-complex.The detection of TNT by SERRS utilising the formation of a Janowskycomplex has been demonstrated previously. This method is advantageous asit is simple and quick and only requires the mixture of the ketone andexplosive in the presence of base.

[0253] In the example reported, acetone was employed to form the enolateanion. SERRS signals were observed, however the ultimate sensitivity ofthe method was dissapointing. The reason for the lack of sensitivity wasattributed to the poor surface adsorption of the complex to the SERRSmetal substrate. Therefore by controlling the surface chemistry involvedit was expected that superior detection limits would be available. Theobvious way to control the surface chemistry is to employ a surfaceseeking ketone.

[0254] Initially, 4-acetylpyridine was chosen as the surface seekingketone. Pyridine compounds are known to adsorb well to Ag⁽¹⁹⁾ andfurthermore this compound is commercially available at low cost. TheJanowsky complex formed with TNT was red/purple in colour with a broadabsorption maximum in the visible (FIG. 19).

[0255] Initial SERRS studies of this complex at 514 nm indicated thatstrong and characteristic signals could be obtained at 10⁻⁵M, equivalentto approximately 10⁻⁸ moles of TNT (FIG. 20). It is expected that byoptimisation of parameters such as aggregation, lower limits ofdetection can easily be achieved. The main feature of the spectrum is abroad band between 1200 and 1400 cm⁻¹, which probably originates fromthe strong nitro stretch of TNT.

[0256] Although the SERRS spectrum obtained from the 4-acetylpyridineJanowsky complex was adequate, better surface chemistry could beachieved with a benzotriazole ketone.

1H-benzotriazole-5-ethanone

[0257] This compound is not commercially available, and hence wassynthesised (see later). A benzotriazole azo ketone (see later) was alsosynthesised to provide a chromophore, surface complexation and reaction.The UV-vis results of this ketone in the presence of TNT and base (FIG.21) show that formation of the TNT/BT azo dye Janowsky complex hasoccurred.

[0258] (ii) Derivatisation of PETN Reduction Product:

[0259] The reduction or hydrolysis of PETN can be achieved readily toafford the mono, di or tri-nitrate ester. Subsequent reaction of theresulting alcohol(s) by a number of procedures is then possible. Forexample, reaction of pentaerythritol with an aldehyde results in thedi-acetal derivative.

[0260] Esterification of the PETN reduction/hydrolysis product(s) isanother method for derivatisation. The reaction of pentaerythritol withisonicotinoyl chloride was achieved to afford the pyridyl ester [4] inquantitative yield. Although this compound was not coloured it wasexpected that the presence of four pyridine rings would enable a strongsurface adsorption at the SERRS metal substrate. Further reaction of thenon-coloured ester with a Cu²⁺ salt produced a coloured complex thatproduced a strong resonance Raman spectrum.

Formation of 1,3-bis-pyridyloxy-2,2-bis-pyridyloxymethyl-propane [4]

[0261] 1,3-Bis-pyridyloxy-2,2-bis-pyridyloxymethyl-propane [4]

[0262] Pentaerythritol (680 mg, 5 mmol) was co-evaporated with anhydrouspyridine (3×25 ml) before dissolving in anhydrous pyridine (50 ml) andcooling to 0° C. in an ice bath. A solution of isonicotinoyl chloride (4g, 20 mmol) in anhydrous pyridine (50 ml) was added to thepentaerythritol solution dropwise with stirring. After addition wascomplete the resulting mixture was refluxed gently for one hour.Pyridine was removed in vacuo to leave an aqueous residue that wasdissolved in ethyl acetate (30 ml) and extracted with sodium chloride(4×20 ml). The organic layer was dried over sodium sulphate and uponremoval of solvent in vacuo, [24] was afforded as a white solid inquantitative yield. R_(f) (A) 0.61; δ_(H)(400 MHZ; DMSO-d₆) 4.76 (8H, s,CH₂) 7.83 (8H, d, Py) 8.74 (8H, d, Py); m/z (FAB) 557.16457 [(M+H)⁺calc. for C₂₉H₂₅N₄O₈ 557.16724].

[0263] (iii) Preparation of Multifunctional Reagents for SERRS

[0264] These molecules have been engineered to contain functionalityreactive to important analyte targets, a chromophore to provideresonance and the ability to complex to the SERRS metal substrate. Thefollowing examples describe the preparation of new benzotriazolederivatives, designed to provide a platform in the synthesis ofimportant SERRS ligands.

[0265] 2,2,2-Trifluoro-N-[1H-benzotriazol-5-yl]-acetamide [17]

[0266] To a stirred solution of 5-amino-1H-benzotriazole (2.68 g, 20mmol) in anhydrous DMF (27 ml) was added dry disopropylethylamine (10ml, 57.4 mmol) and trifluoroacetic anhydride (TFAA) (20 ml). Stirringwas continued at room temperature overnight, by which time TLC (A) andninhydrin development indicated the conversion of starting material. DMFand excess TFAA were remove in vacuo to afford an aqueous residue thatwas dissolved in ethyl acetate (50 ml) and extracted with sodiumchloride solution (3×50 ml). The organic layer was dried over sodiumsulphate and then removed in vacuo to afford pure [17] as an off whitesolid (5.44 g, 84%). R_(f) (A) 0.64; δ_(H)(400 MHz; DMSO-d₆) 7.63 (1H,d, J8.9) 7.98 (1H, d, J8.8) 8.30 (1H, s) 11.49 (1H, s, NH); m/z (EI−HR)230.04154 [(M+H)⁺ calc. for C₈H₅N₄OF₃ 230.04155].

[0267] N-(1-Acetyl-1H-benzotriazol-5-yl)-acetamide [18]

[0268] A solution of 5-amino-1H-benzotriazole (1.07 g, 8 mmol) in aceticanhydride (50 ml, excess) was stirred at 90° C. for two hours, by whichtime TLC (A) and ninhydrin development indicated conversion of startingmaterials. Excess acetic anhydride and acetic acid were removed in vacuoto afford [18] as an off white solid. R_(f) (A) 0.48; m/z (FAB)219.08632 [(M+H)⁺ calc. for C₁₀H₁₁N₄O₂ 219.08820].

[0269] 5,6-Dinitro-1H-benzotriazole [19]

[0270] 5-Nitro-1H-benzotriazole was dissolved in conc. sulphuric acid(60 ml) and cooled to 0° C. in an ice bath. Nitric acid (60 ml, excess)was added drop wise to the cooled solution over a period of 20 minutes.Stirring was continued for another 15 minutes at 0° C. and then at 115°C. overnight, by which time TLC (A) indicated the conversion of startingmaterial. The solution was cooled to room temperature and poured overice to precipitate a pale yellow solid and a clear yellow solution. Thesolid was collected by filtration and washed with copious amounts ofwater until neutral to afford [19] in 67% yield. R_(f) (A) 0.34;δ_(H)(400 MHZ; DMSO-d₆) 9.02 (2H, s, BT); m/z (FAB) 210.02695 [(M+H)⁺calc. for C₆H₄N₅O₄ 210.02633].

[0271] 5,7-Dinitro-1H-benzotriazole [20]

[0272] The clear yellow solution obtained after precipitation of[19] wasneutralised and extracted with ethyl acetate (50 ml) and sodium chloridesolution (4×20 ml) to afford a yellow organic layer that was dried oversodium sulphate. Removal of the solvent in vacuo afforded [20] as astraw coloured solid in 30% yield. R_(f) (A) 0.48; δ_(H)(400 MHZ;DMSO-d₆) 9.02 (1H, s, BT) 9.54 (1H, s, BT); m/z (FAB) 210.02545 [(M+H)⁺calc. for C₆H₄N₅O₄ 210.02633].

[0273] 7-Nitro-1H-benzotriazole-5-carboxylic acid [21]

[0274] 1H-Benzotriazole-5-carboxylic acid (1 g, 6 mmol) was dissolved,in conc. sulphuric acid (20 ml) and cooled to 0° C. on an ice bath.Nitric acid (20 ml, excess) was added drop wise to the cooled solutionover a period of 20 minutes. Stirring was continued for another 15minutes at 0° C. and then at 90° C. for 2 hours, by which time TLC (A)indicated the conversion of starting material. The solution was cooledto room temperature and poured over ice to precipitate a white solid.The solid was collected by filtration and washed with copious amounts ofwater until neutral to afford [21] in 48% yield. R_(f) (A) 0.53;δ_(H)(400 MHz; DMSO-d₆) 8.77 (1H, s, BT) 9.01 (1H, s, BT) 13.75 (1H, brs, OH) 16.90 (1H, s, NH); m/z (EI−HR) 208.02288 [(M) calc. for C₇H₄N₄O₄208.02325].

[0275] 4-Nitro-5-chloro-1H-benzotriazole [22]

[0276] 5-Chloro-1H-benzotriazole (3.08 g, 20 mmol) was dissolved inconc. sulphuric acid (40 ml) and cooled to 0° C. on an ice bath. Nitricacid (40 ml, excess) was added drop wise to the cooled solution over aperiod of 20 minutes. Stirring was continued for 1 hour at 0° C. andthen at 60° C. for an additional 1 hour, by which time TLC (A) indicatedthe conversion of starting material. The solution was cooled to roomtemperature and poured over ice to precipitate a white solid. The solidwas collected by filtration and washed with copious amounts of wateruntil neutral to afford [22] in 83% yield. R_(f) (A) 0.49 δ_(H)(400 MHz;DMSO-d₆) 7.75 (1H, d, J8.8, BT) 8.38 (1H, br s, BT); m/z (EI−HR)197.99383 [(M) calc. for C₆H₃N₄O₂ ³⁵Cl 197.99445] and 199.99345 [(M)calc. for C₆H₃N₄O₂ ³⁷Cl 199.99150].

[0277] 4-Nitro-5-methyl-1H-benzotriazole [23]

[0278] Nitric acid (40 ml, excess) was added drop wise over 20 minutesto a stirred solution of 5-methyl-1H-benzotriazole (2.66 g, 20 mmol) inconc. sulphuric acid (40 ml) at 0° C. Stirring was continued at roomtemperature for 90 minutes by which time TLC (A) indicated completereaction. The solution was poured onto ice to precipitate a flocculentwhite solid. The solid was collected by filtration and washed with waterto afford [23] in 94% yield. R_(f) (A) 0.60 δ_(H)(400 MHz; DMSO-d₆) 2.79(3H, s, CH₃) 7.52 (1H, d, J8.4, BT) 8.36 (1H, br s, BT) 16.26 (1H, s,NH); m/z (EI−HR) 178.04937 [(M) calc. for C₇H₆N₄O₂ 178.04908].

[0279] 1H-Benzotriazole-5-carboxylic acid ethyl ester [24]

[0280] A suspension of 1H-benzotriazole-5-carboxylic acid in ethanol (50ml, excess) was gently refluxed overnight in the presence of conc.sulphuric acid (0.5 ml), by which time TLC (A) indicated completereaction. Ethanol was removed in vacuo to afford an aqueous residue thatwas dissolved in ethyl acetate (30 ml) and extracted with sodiumchloride solution (4×20 ml). The organic layer was dried over sodiumsulphate and removal of solvent in vacuo afforded the pure ester [24] in86% yield. R_(f) (A) 0.75; δ_(H)(400 MHz; DMSO-d₆) 1.36 (3H, t, CH₃)4.36 (2H, q, CH₂) 7.94 (2H, br s, BT) 8.59 (1H, br s, BT) 16.03 (1H, s,NH); m/z (EI−HR) 191.07023 [(M) calc. for C₉H₉N₃O₂ 191.06948].

[0281] 5-Acetyl-1H-Benzotriazole [25]

[0282] The ketone was prepared in the same manner as the benzotriazolealdehyde from 5-aminobenzotriazole and acetaldoxime. Columnchromatography of the crude ketone using DCM and eluting with MeOH(0-10%) afforded the ketone in 11% yield. R_(f) [A] 0.3; δ_(H)(400 MHz;Acetone-d₆) 2.68 (3H, s, CH₃), 7.92 (1H, d, J 8.7, Ar—H), 8.07 (1H, d, 38.7 Ar—H), 8.62 (1H, s, Ar—H); m/z (FAB−HR) 162.06674 [(M+H)⁺ calc. forC₈H₈N₃O 162.06682]. The hydrazone of the ketone was prepared from2,4DNPH to afford an orange solid.

[0283] 4,6-Dinitro-7-1H-benzotriazole [26]

[0284] 1H-benzotriazole (5 g) was dissolved in conc. sulphuric acid (60ml) and cooled to 0° C. in an ice bath. Nitric acid (60 ml, excess) wasadded drop wise to the cooled solution over a period of 20 minutes.Stirring was continued for another 15 minutes at 0° C. and then at 120°C. for 48 hrs, by which time TLC (A) indicated the conversion ofstarting material. The solution was cooled to room temperature andpoured over ice to precipitate a white solid and leave a clear yellowsolution. The solid was collected by filtration and washed with copiousamounts of water until neutral to afford [26] in 46% yield.

[0285] 4,7-Dinitro-1H-benzotriazole [27]

[0286] The clear yellow solution obtained after precipitation of [26]was neutralised and extracted with ethyl acetate (50 ml) and sodiumchloride solution (4×20 ml) to afford a yellow organic layer that wasdried over sodium sulphate. Removal of the solvent in vacuo afforded astraw coloured solid containing 4 nitro BT, 4,6 dinitro BT and [27] in acrude yield of 35%. Column chromatography in hexane, eluting with ethylacetate (0-30%) afforded the title compound.

[0287] 5-Trifluoromethyl-1H-benzotriazole [28]

[0288] 1-Trifluromethyl-3,4-phenylenediamine (1.056 g, 6 mmol) wasdissolved in acetic acid (15 ml) containing concentrated HCl (0.7 ml).This resulted in the precipitation of a white solid (the HCl salt of theamine). The suspension was cooled to 0° C. in an ice bath and NaNO₂(0.455 g, 1.1 eq) in water (5 ml) was added dropwise over 20 minutes.The suspension dissolved to afford a clear dark solution of thediazonium salt. Stirring was continued at 0° C. for 15 minutes and thenat room temperature for a further 15 minutes to afford an orange/yellowsolution. The aqueous solution was extracted with EtOAc (50 ml) andNaCl. The organic layer was kept and the solvent removed to give anacidic residue. Co-evaporation with toluene gave [28] as a tan solid inquantitative yield.

[0289] 6-Nitro-5-trifluoromethyl-1H-benzotriazole [29] and7-Nitro-5-trifluoromethyl-1H-benzotriazole [30]

[0290] Compound [28] (0.935 g) was dissolved in conc. sulphuric acid (10ml) and cooled to 0° C. in an ice bath. Nitric acid (10 ml) was addeddrop wise to the cooled solution over a period of 20 minutes. Stirringwas continued for another 15 minutes at 0° C. and then at 100° C. for 2hrs, by which time TLC (A) indicated the conversion of startingmaterial. The solution was cooled to room temperature and poured overice to precipitate a white solid and leave a clear yellow-solution. Thesolid was collected by filtration and washed with copious amounts ofwater until neutral to afford a mixture of [29] and [30]. The aqueousfiltrate was extracted to give a further quantity of the mixture. Theoverall yield of the nitration was 66%. Compound [29] was obtained in62% and compound [30] in 38%. The two nitro derivatives were separatedby chromatography using hexane and eluting with ethyl acetate and thenmethanol to afford the pure isomers.

[0291] Selective Reduction of Dinitrobenzotriazoles to Nitro AminoBenzotriazole

[0292] The dinitrobenzotriazole was dissolved in acetic acid and heatedto 70° C. Fe (3 eq) was then added to the solution in one portion andstirring was continued until the reaction was complete by TLC. Theacidic solution was then extracted with EtOAc/NaCl to leave an acidicorganic layer. The solvent was removed to give an acidic residue thatwas co-evaporated with toluene to afford the nitro amino derivative inmoderate to high yield.

[0293] Other Important SERRS Based Synthons

[0294] The following synthetically and analytically useful reagents havealso/can also be prepared. Two other methods that can be utilised forthe preparation of a benzotriazole aldehyde are also presented.

[0295] wherein R above has the same definitions as X herein beforedescribed.

EXAMPLE 10 Preparation of Colloid by Batch Process and Flow Cell System

[0296] a) Batch Process

[0297] A glass coil was designed to produce borohydride-reduced colloidin batches by pumping solutions of silver nitrate and sodium borohydridethrough the coil. A diagram of this system is shown in FIG. 17. Thesolutions were pumped through PVC tubing using a peristaltic pump intotwo glass inlet arms. The inlet arms then join at a point in the coilwhere there are indentations in the glass to promote mixing. Thecolloid, which has formed at this point, is pumped through a coil whichis immersed in either a water bath or an ice bath. At the other end ofthe coil there is another inlet arm through which a solution oftrisodium citrate can be introduced. The colloid then pumps out into acollecting vessel. The overall flow rate can be changed by altering thespeed of the peristaltic pump and the relative flow rates of each of thesolutions can be altered by changing the diameter of the tubing used.

[0298] Sample Preparation

[0299] Fresh solutions of sodium borohydride, silver nitrate andtrisodium citrate were used for each colloid preparation. Prior tocolloid preparation the system was cleaned with 20% nitric acid andrinsed thoroughly with distilled water. Each of the colloids preparedusing the batch flow system were tested using SERRS and UV-Vis. Sampleswere prepared as follows:

[0300] SERRS

[0301] 2 cm³ of colloid was mixed with 50 μL of GM19 (Graham, D.;McLauglin, C.; McAnally, G.; Jones, J. C.; White, P. C.; Smith, W. E.Chemical Communications, 1998, 1187-1188) (10⁻⁶ mol dm⁻³) in a cuvetteand left for 5 minutes to allow the dye to attach to the surface. Thiswas then mixed with 50 μL sodium chloride (1 mol dm⁻³). Methanol spectrawere run in between each sample and the intensities of the GM19 spectrawere normalised against the average intensity of the methanol peak atapproximately 1035 cm⁻¹ in the spectra run before and after each sample.

[0302] UV-Visible Spectroscopy

[0303] A background spectrum of distilled water was run prior to thesamples. The spectra from 300 to 800 mn were obtained from undilutedsamples of the colloid.

[0304] b) Flow System in Line with Flow Cell

[0305] A small flow system for colloid preparation was used in line witha flow cell to produce a SERRS signal from an analyte immediately afterthe colloid was made. Silver nitrate and sodium borohydride solutionswere pumped into a small coil where the colloid was produced. This wasconnected to a flow cell. The colloid then flowed through one coilbefore it was mixed with an aggregating agent which was pumped inthrough an inlet. This was then pumped through another coil before beingmixed with the analyte which was pumped in through a further inlet. Thiswas then pumped through a capillary and the Raman scattering from theanalyte was accumulated from the capillary.

[0306] Sample Preparation

[0307] Three dyes were used to test the effectiveness of this system.GM19, which was used to test the batch flow system,5-(5′-azobenzotriazole)8-hydroxyquinoline (EP-96-1), and4-(5′-benzotriazole)-2,4-dinitrophenylhydrazone.

[0308] Aqueous solutions of the dyes (10⁻⁶ mol dm ³) were pumped throughthe system. The tubing diameters for all solutions were the same,therefore the dye was diluted a further three times prior to analysisgiving a final concentration of 2.5×10⁻⁷ mol dm⁻³ at the point ofanalysis. The aggregating agents used were NaCl (1 mol dm⁻³) andpoly-1-lysine (0.01%).

[0309] As it is reported in the literature that citrate-reduced silvercolloids are stable for longer periods of time than borohydride-reducedcolloids it was thought that adding citrate to borohydride colloids mayincrease their stability.

[0310] Five 10 cm³ aliquots of borohydride colloid 5 were measured outinto glass vials and different amounts of citrate were added to eachone. The amount of citrate added to aliquot 1 was equivalent to theamount used to prepare the citrate-reduced colloids and the amountsadded to each subsequent aliquot were calculated by halving the amountadded to the previous aliquot.

[0311] The UV-Vis spectrum of each aliquot was run after the citrate wasadded and every week for one month. Three months after preparation, whenmost of the borohydride colloids had aggregated, the UV-Vis spectra ofthe citrate-stabilished borohydride aliquots were run. ANOVA was carriedout for the position of the peak maximum and the absorbance from theUV-Vis data and this is shown in Table 1.

[0312] Table 1: ANOVA of the Position of the Peak Maximum from theUV-Vis Spectra of the Citrate-stabilised Colloids Monitored over a ThreeMonth Period. TIME ALI- ALI- ALI- ALI- ALI- (WEEKS) QUOT 1 QUOT 2 QUOT 3QUOT 4 QUOT 5 0 0.7191 0.705 0.7225 0.7346 0.7178 1 0.7265 0.7083 0.73330.7426 0.731 2 0.7178 0.7053 0.7328 0.7335 0.7334 3 0.7248 0.7024 0.74570.7358 0.7484 4 0.7455 0.7018 0.7356 0.7417 0.6972 12 0.716 0.69030.7334 0.7174 0.7026

[0313] Studies were carried out to investigate the reproducibility ofmaking colloid by a conventional batch process and the new flow cellprocess. For citrate reduced silver colloid prepared by a batch process,the percentage RSD for 5 batches was 38% and for borohydride reducedsilver colloid prepared by a batch process, the percentage RSD for 5batches was 41%. However the percentage RSD for 8 batches of colloidproduced by the new flow cell process was 22.9% showing that thereproducibility of the flow cell process is better that the batchprocess.

EXAMPLE 11 TNT Detection Using Azo-derivatisation Chemistry in a FlowCell Apparatus

[0314] Prior to passing a sample of TNT through the flow cell apparatusshown in FIG. 17, the TNT must first be captured and transferred tosolution. Vapour from explosive materials present in the atmosphere istrapped on ‘Tenax’ (adsorbent polymeric material). The adsorbed TNT isthen desorbed from the Tenax by washing with acetic acid.

[0315] The sampling tube consists of Tenax supported with glass wool ina glass tube through which air is drawn using a pump. The tube can thenbe connected to the flow system where the TNT is washed off the Tenaxand derivatised.

[0316]FIG. 23 shows a diagram of the flow cell apparatus used for SERRSdetection of the azo derivatised TNT. The derivatisation of the TNT,preparation of the SERRS substrate (borohydride-reduced silver colloid),attachment of the derivatised dye to the silver surface and subsequentdetection of the azo derivatised TNT are all carried out on-line.

[0317] The reaction scheme for the formation of5-(2-methyl-3,5-dinitro-phenylazo) quinolin-8-ol is shown in FIG. 24.

[0318]FIG. 25 shows a SERRS spectrum of the product formed in the flowsystem from 1.1×10⁻⁸ g TNT. The spectrum was recorded in 10 s using514.5 run excitation.

[0319] Derivatisation of TNT in the flow cell is carried out as follows.The TNT is washed off the Tenax tube (7) using acetic acid, reduced andthen coupled with 8-hydroxyquinoline to form5-(2-methyl-3,5-dinitro-phenylazo) quinolin-8-ol. The reduction of TNTis carried out by passing the solution of TNT in acetic acid through aglass tube containing iron powder (20 mg) (8) which is held in placewith plugs of glass wool. This tube is connected to line (1) of the flowsystem. While the TNT is flowing through the glass reduction tube, it isheated to 90° C. in a copper block (9). The resulting reduced TNT isthen collected on a column packed with Amberlite CG-120 (10). Thisenables the separation of the reduced TNT from any excess iron producedduring the reduction stage. The reduced TNT is then washed off thecolumn using acetone. Sodium nitrite solution (2.7×10⁻⁴ M in 10% H₂SO₄)is pumped in through inlet (2). The diazotisation occurs at point (11),where the flow cell is cooled to between 0 and 5° C. using peltiercoolers (11) which are attached to a copper block through which the flowcell passes. 8-Hydroxyquinoline (1×10⁻⁴ M in 1M sodium acetate andacetone), is introduced through inlet (3). At this point the dye,5-(2-methyl-3,5-dinitro-phenylazo) quinolin-8-ol is formed. Sodiumhydroxide (3.4M in distilled water) is pumped in through inlet (4) toadjust the pH of the solution. Colloid is prepared by introducing sodiumborohydride solution (1.1×10−³M in 0.1M sodium hydroxide solution)through inlet (5) and silver nitrate solution (2.6×10⁻³M in distilledwater) through inlet (6). The silver colloid and dye then mix at point(12) and the SERRS spectra are accumulated form the solutions as theypass through the capillary (14). The flow rates of reagents were asfollows. Inlets (1), (2), (3), and (6) run at 0.7 mL min⁻¹, inlet (4)runs at 1.00 mL min⁻¹ and inlet (5) runs at 11.2 mL min.

1. A method for detecting an analyte in a sample using surface enhanced(resonance) Raman scattering (SE(R)RS) detection, comprising the stepsof a) mixing the sample with a reagent such that any analyte present inthe sample reacts with the reagent thereby forming a derivatisedanalyte, wherein the derivatised analyte comprises a chromophore; b)mixing said derivatised analyte with a SE(R)RS active substrate so as toadhere the derivatised analyte thereto; and c) detecting the derivatisedanalyte by way of SE(R)RS detection whereby any derivatised analytedetected may be correlated with analyte present in the sample.
 2. Themethod according to claim 1 wherein SERRS detection is employed.
 3. Themethod according to claim 1 wherein the analyte is selected from thegroup consisting of aldehydes, amines, explosives, drugs of abuse,therapeutic agents, metabolites and environmental pollutants andbiological samples such as antibodies, proteins, lipids, nucleic acids,polypeptides, polyketides and glycosides.
 4. The method according toclaim 3 wherein the analyte is an explosive and is selected from TNT,RDX and PETN.
 5. The method according to claim 1 wherein the analyte tobe detected is in the vapour phase which is collected and dissolved in asolvent prior to mixing with the reagent.
 6. The method according toclaim 1 wherein the analyte is chemically functionalised by reduction,hydrolysation and/or oxidation prior to reacting with the analyte. 7.The method according to claim 1 wherein the reagent which is used toderivatise the analyte or functionalised analyte, provides achromophore; provides in combination with the analyte or functionalisedanalyte a chromophore; and/or renders the analyte susceptible toadhering to the SE(R)RS active substrate.
 8. The method according toclaim 7 wherein the reagent derivatises the analyte in order to providea chromophore and the derivatised analyte is adhered to the SE(R)RSactive substrate by way of an aggregating agent.
 9. The method accordingto claim 8 wherein the aggregating agent is poly-L-lysine.
 10. Themethod according to claim 7 wherein the analyte is reacted with thereagent according to formula I:

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxyl amine, haloalkyl, hydroxyl, or otheractive group.
 11. The method according to claim 10 wherein X is selectedfrom the group consisting of, —NH₂, —R—CONH₂, —CHO, NO, NHOH, CF₃ andH₂NCO—R—CONH₂ wherein R is C₁-C₄ alkyl or alkenyl, a diazonium halide,or a mono, di or tri nitro phenyl.
 12. The method according to claim 7wherein the analyte is reacted with a reagent according to either offormulae II or III

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxyl amine, haloalkyl, hydroxyl, or otheractive group and Y is an alkyl, aryl, alkenyl, alkynyl, cycloalkyl groupincluding derivatives of the preceding groups, or Y can be any atom thatcan provide two or more bonds to link the two groups together such as Oor B.
 13. The method according to claim 12 wherein Y is an amine, imineor azo linkage.
 14. The method according to claim 7 wherein the analyteis reacted with a reagent according to formula IV:

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxl amine, hydroxyalkyl, hydroxy, or otheractive group.
 15. The method according to claim 1 wherein the reactionsand detection are carried out in situ.
 16. The method according to claim15 wherein the reactions and detection are carried out in a singleapparatus.
 17. The method according to either of claims 15 or 16 whereinthe time taken from obtaining the sample to generating a SE(R)RSspectrum is less than 1 minute.
 18. The method according to claim 15wherein the reactions are carried out in a apparatus comprising a flowcell for mixing and allowing reaction of the various components.
 19. Themethod according to claim 1 wherein the SE(R)RS active substrate is aroughened metallic surface, a metal sol or, an aggregation of metalcolloid particles.
 20. The method according to claim 19 wherein themetal surface is coated with citrate, polylysine or polyphenol.
 21. Themethod according to claim 19 wherein the SE(R)RS active substrate is anaggregation of metal colloid particles which have been prepared bymixing a suitable reducing agent with metal nitrate and aggregating thecolloid particles by mixing with an aggregating agent is a flow cell.22. The method according to claim 21 wherein the aggregating agent is anacid, polyamine or inorganic activating ion.
 23. The method according toclaim 22 wherein the preparation of colloid particles is carried out insitu in the same apparatus as the reactions and detection of theanalyte.
 24. The method according to claim 1 wherein the derivatisedanalyte is adhered to the SE(R)RS-active surface by chemi-sorption, orchemical bonding.
 25. A detection device for detecting the presence ofan analyte in a sample according to the method according to anypreceding claim, the device comprising at least one flow cell forcombining in situ the sample to be analysed, a reagent capable ofreacting with any analyte present in the sample in order to provide aderivatised analyte comprising a chromophore, and thereafter reactingwith a SE(R)RS active substrate so as to adhere the derivatised analytethereto and detecting the derivatised analyte by way of SE(R)RS.
 26. Thedetection device according to claim 24 wherein the SE(R)RS activesubstrate is prepared in the device using a flow cell, prior to reactingwith the derivatised analyte.
 27. A method of preparing a colloid foruse in SE(R)RS analysis, comprising the steps of mixing a metal nitratewith a reducing agent using a flow cell in order to form a colloid andthereafter adding citrate in order to stabilise the colloid.
 28. Acompound according to formula I for use in SE(R)RS analysis:

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxyl amine, haloalkyl, hydroxyl, or otheractive group.
 29. A compound according to either of formulae II or IIIfor use in SE(R)RS analysis:

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxyl amine, haloalkyl, hydroxyl, or otheractive group and Y is an alkyl, aryl, alkenyl, alkynyl, cycloalkyl groupincluding derivatives of the preceding groups, or Y can be any atom thatcan provide two or more bonds to link the two groups together such as Oor B.
 30. A compound according to formula IV for use in SE(R)RSanalysis:

wherein X may be substituted on one or more positions of the aromaticring and is an amine, amide, aldehyde, thiol, diazo group, nitro, avinyl group, nitroso, hydroxl amine, hydroxyalkyl, hydroxy, or otheractive group.